Tumor necrosis factor-gamma

ABSTRACT

Human TNF-gamma-alpha and TNF-gamma-beta polypeptides and DNA (RNA) encoding such polypeptides and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptides to inhibit cellular growth, for example in a tumor or cancer, for facilitating wound-healing, to provide resistance against infection, induce inflammatory activities, and stimulating the growth of certain cell types to treat diseases, for example restenosis. Also disclosed are diagnostic methods for detecting a mutation in the TNF-gamma-alpha and TNF-gamma-beta nucleic acid sequences or overexperession of the TNF-gamma-alpha and TNF-gamma-beta polypeptides. Antagonists against such polypeptides and their use as a therapeutic to treat cachexia, septic shock, cerebral malaria, inflammation, arthritis and graft-rejection are also disclosed.

This application claims benefit under 35 U.S.C. § 119(e) of the filingdate of U.S. Provisional Application Serial No. 60/074,047, filed onFeb. 9, 1998. Further, this application claims benefit under 35 U.S.C. §120 as a continuation-in-part of U.S. application Ser. No. 09/131,237,filed on Aug. 7, 1998, which is a continuation-in-part of U.S.application Ser. No. 09/005,020, filed on Jan. 9, 1998, abandoned, whichis a continuation-in-part of U.S. application Ser. No. 08/461,246, filedon Jun. 5, 1995, abandoned, which is a continuation-in-part ofInternational Application Serial No. PCT/US94/12880, filed on Nov. 7,1994. Each of the five aforementioned applications are herebyincorporated by reference in their entireties.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides. More particularly, the polypeptide of the presentinvention has been identified as a new member of the tumor necrosisfactor family and is hereinafter referred to as “TNF-gamma-alpha”. Theinvention also relates to a protein encoded by a splice variant of thegene encoding TNF-gamma-alpha which is hereinafter referred to as“TNF-gamma-beta”. The invention also relates to inhibiting the action ofsuch polypeptides.

BACKGROUND OF THE INVENTION

Human tumor necrosis factors—alpha (TNF-alpha) and beta (TNF-beta orlymphotoxin)—are related members of a broad class of polypeptidemediators, which includes the interferons, interleukins and growthfactors, collectively called cytokines (Beutler, B. and Cerami, A.,Annu. Rev. Immunol., 7:625-655 (1989)).

Tumor, necrosis factor (TNF-a and TNF-b) was originally discovered as aresult of its anti-tumor activity, however, now it is recognized as apleiotropic cytokine playing important roles in immune regulation andinflammation, To date, there are eight known members of the TNF-relatedcytokine family, TNF-alpha, TNF-beta, (lymphotoxia (LT)-alpha, LT-beta,and ligands for the Fas, CD30, CD27, CD40 and 4-1BB receptors. Theseproteins have conserved C-terminal sequences and variable N-terminalsequences which are often used as membrane anchors, with the exceptionof TNF-beta. Both TNF-alpha and TNF-beta function as homotrimers whenthey bind to TNF receptors.

TNF is produced by a number of cell types, including monocytes,fibroblasts, T-cells, natural killer (NK) cells and predominately byactivated machrophages. TNF-alpha has been reported to have a role inthe rapid necrosis of tumors, immunostimulation, autoimmune disease,graft rejection, resistance to parasites, producing an anti-viralresponse, septic shock, growth regulation, vascular endothelium effectsand metabolic effects. TNF-alpha also triggers cells to secrete variousfactors, including PAI-1, IL-1, GM-CSF and IL-6 to promote cellproliferation. In addition, TNF-alpha regulates various cell adhesionmolecules such as E-Selectin, ICAM-1 and VCAM-1. TNF-alpha and Fasligand have also been shown to induce programmed cell death.

The first step in the induction of the various cellular responsesmediated by TNF or LT is their binding to specific cell surfacereceptors. Two distinct TNF receptors of approximately 55-KDa (TNF-R1)and 75-KDa (TNF-R2) have been identified (Hohman, H. P. et al., J. Biol.Chem., 264:14927-14934 (1989)), and human and mouse cDNAs correspondingto both receptor types have been isolated and characterized (Loetscher,H. et al., Cell, 61:351 (1990)). Both TNF-Rs share the typical structureof cell surface receptors including extracellular, transmembrane andintracellular regions.

The endothelium, which under physiological conditions is mostly aquiescent tissue (Denekamp, J. Cancer Metas. Rev. 9:267-282 (1990)),plays an essential role in the maintenance of vascular homeostasis andpermeability. Endothelial cells are actively involved in inflammation,cell adhesion, coagulation, thrombosis, fibrinolysis, and angiogenesis.During angiogenesis, endothelial cells proliferate, invade into stroma,migrate toward the source of an angiogenesis stimulus, such as cancercells, interact with perivascular cells and stromal cells, andeventually, form capillary vessels linking the tumor tissue to thecirculatory system (Folkman, J. Nature Med. 1:27-31 (1995)). Althoughthe complex mechansim that regulates angiogenesis is yet to be fullyunderstood, it is becoming clear that the initiation or termination ofthe process is a result of a balance between positive and negativefactors.

A number of angiogenic factors, often markedly upregulated in tumortissues, have been described. These include several members of thefibroblast growth factor (FGF) family, such as FGF-1, FGF-2, and thoseof the vascular endothelial cell growth factor (VEGF) family and thereceptors for all of these molecules (Gimenez-Gallego, G, et al.,Science 230:1385-1388 (1985); Schweigerer, L., et al., Nature325:257-259 (1987): Leung, D. W., et al., Science 246:1306-1309 (1989);Burrus, L. W. and Olwin, B. B. J. Biol. Chem. 264:18647-18653 (1989);Wennstrom, S., et al., Growth Factors 4:197-208 (1991); Terman, B. I.,et al., Biochem. Biophys. Res. Comm. 187:1579-1586 (1992); de Vries, C.,et al., Science 255:989-991 (1992)). Likewise, several inhibitors ofangiogenesis have also been reported, including thrombospondin,angiostatin, endostatin, and platelet factor-4 (Good, D. J., et al.,Proc. Natl. Acad. Sci. USA 87:6623-6628 (1990); O'Reilly, M. S., et al.,Cell 79:315-328 (1994); O'Reilly, M. S., et al., Cell 88:277-285 (1997);Maione, T. E., et al., Science 247:77-79 (1990)). It is apparent thatnormal angiogenesis is promptly activated when needed, and swiftlyterminated when no longer required. However, pathological angiogenesis,once initiated, is often prolonged and often difficult to stop. This mayindicate that a negative regulatory mechanism normally functioning ismissing or suppressed in a pathological angiogenic process. It isconceivable that endothelial cells may produce autocrine factors tosuppress an angiogenesis process or maintain the quiescence of a maturevasculature.

The polypeptide of the present invention has been identified as a novelmember of the TNF family based on structural, amino acid sequencehomology, and functional similarities, for example, TNF-gamma is apro-inflammatory protein. Further, the TNF-gamma polypeptide of thepresent invention is a negative regulator of angiogenesis and ofendothelial cell growth. There is a need for polypeptides that functionin this manner, since disturbances of such regulation may be involved indisorders relating to angiogenesis, hemostasis, tumor metastisis,cellular migration, and cancers of many systems. Therefore, there is aneed for identification and characterization of such human polypeptideswhich can play a role in detecting, preventing, ameliorating orcorrecting such disorders.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, there isprovided a novel mature polypeptide which is TNF-gamma-alpha, and anovel mature polypeptide which is TNF-gamma-beta, as well asbiologically active and diagnostically or therapeutically usefulfragments, analogs and derivatives thereof.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules encoding human TNF-gamma-alphaor TNF-gamma-beta, including mRNAs, DNAs, cDNAs, genomic DNAs as well asanalogs and biologically active and diagnostically or therapeuticallyuseful fragments and derivatives thereof.

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding at least a portion of theTNF-gamma-alpha polypeptide having the complete amino acid sequenceshown in SEQ ID NO:2 or the complete amino acid sequence encoded by thecDNA clone HUVEO91 deposited as plasmid DNA as ATCC Deposit Number 75927at the American Type Culture Collection (“ATCC”) on Oct. 26, 1994. TheATCC is located at 10801 University Boulevard, Manassas, Va. 20110-2209,USA. The nucleotide sequence determined by sequencing the depositedTNF-gamma-alpha clone, which is shown in FIGS. 1A-C (SEQ ID NO:1),contains an open reading frame encoding a complete polypeptide of 174amino acid residues, including an initiation codon encoding anN-terminal methionine at nucleotide positions 783-785, and a predictedmolecular weight of about 20,132 Da.

The present invention also provides isolated nucleic acid moleculescomprising a polynucleotide encoding at least a portion of theTNF-gamma-beta polypeptide having the complete amino acid sequence shownin SEQ ID NO:20 or the complete amino acid sequence encoded by the cDNAclone HEMCZ56 deposited as plasmid DNA as ATCC Deposit Number 203055 onJul. 9, 1998. The nucleotide sequence determined by sequencing thedeposited TNF-gamma-beta clone, which is shown in FIGS. 20A and B (SEQID NO:20), contains an open reading frame encoding a completepolypeptide of 251 amino acid residues, including an initiation codonencoding an N-terminal methionine at nucleotide positions 1-3, and apredicted molecular weight of about 28,089 Da.

Thus, in one embodiment the invention provides an isolated nucleic acidmolecule comprising a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding the TNF-gamma-alpha polypeptide having the complete amino acidsequence in SEQ ID NO:2 (i.e., positions −27 to 147 of SEQ ID NO:2, (b)a nucleotide sequence encoding the TNF-gamma-alpha polypeptide havingthe complete amino acid sequence in SEQ ID NO:2 excepting the N-terminalmethionine (i.e., positions −26 to 147 of SEQ ID NO:2); (c) a nucleotidesequence encoding the mature TNF-gamma-alpha polypeptide having theamino acid sequence in SEQ ID NO:2 shown as positions 1 to 147 of SEQ IDNO:2; (d) a nucleotide sequence encoding the TNF-gamma-alpha polypeptidehaving the complete amino acid sequence encoded by the cDNA cloneHUVEO91 contained in ATCC Deposit No. 75927; (e) a nucleotide sequenceencoding the TNF-gamma-alpha polypeptide having the complete amino acidsequence excepting the N-terminal methionine encoded by the cDNA cloneHUVEO91 contained in ATCC Deposit No. 75927; (f) a nucleotide sequenceencoding the mature TNF-gamma-alpha polypeptide having the amino acidsequence encoded by the cDNA clone HUVEO91 contained in ATCC Deposit No.75927; and (g) a nucleotide sequence complementary to any of thenucleotide sequences in (a), (b), (c), (d), (e) or (f), above.

In another embodiment, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding the TNF-gamma-beta polypeptide having the complete amino acidsequence in SEQ ID NO:20 (i.e., positions 1 to 251 of SEQ ID NO:20); (b)a nucleotide sequence encoding the TNF-gamma-beta polypeptide having thecomplete amino acid sequence in SEQ ID NO:20 excepting the N-terminalmethionine (i.e., positions 2 to 251 of SEQ ID NO:20); (c) a nucleotidesequence encoding the mature TNF-gamma-beta polypeptide having the aminoacid sequence in SEQ ID NO:20 shown as positions 62 to 251 of SEQ IDNO:20; (d) a nucleotide sequence encoding the TNF-gamma-beta polypeptidehaving the complete amino acid encoded by the cDNA clone HEMCZ56contained in ATCC Deposit No. 203055; (e) a nucleotide sequence encodingthe TNF-gamma-alpha polypeptide having the complete amino acid sequenceexcepting the N-terminal methionine encoded by the cDNA clone HEMCZ56contained in ATCC Deposit No. 203055; (f) a nucleotide sequence encodingthe mature TNF-gamma-beta polypeptide having the amino acid sequenceencoded by the cDNA clone HEMCZ56 contained in ATCC Deposit No. 203055;and (g) a nucleotide sequence complementary to any of the nucleotidesequences in (a), (b), (c), (d), (e) or (f), above.

Further embodiments of the invention include isolated nucleic acidmolecules that comprise a polynucleotide having a nucleotide sequence atleast 90% identical, and more preferably at least 95%, 96%, 97%, 98% or99% identical, to any of the nucleotide sequences in (a), (b), (c), (d),(e), (f) or (g), above, or a polynucleotide which hybridizes understringent hybridization conditions to a polynucleotide in (a), (b), (c),(d), (e), (f) or (g), above, a fragment thereof (such as, for example,fragments described herein), or the complementary strand thereto. Thispolynucleotide which hybridizes does not hybridize under stringenthybridization conditions to a polynucleotide having a nucleotidesequence consisting of only A residues or of only T residues. Anadditional nucleic acid embodiment of the invention relates to anisolated nucleic acid molecule comprising a polynucleotide which encodesthe amino acid sequence of an epitope-bearing portion (i.e., a fragment)of a TNF-gamma polypeptide having an amino acid sequence in (a), (b),(c), (d), (e) or (f), above. A further embodiment of the inventionrelates to an isolated nucleic acid molecule comprising a polynucleotidewhich encodes the amino acid sequence of a TNF-gamma polypeptide havingan amino acid sequence which contains at least one amino acidsubstitution, but not more than 50 amino acid substitutions, even morepreferably, not more than 40 amino acid substitutions, still morepreferably, not more than 30 amino acid substitutions, and still evenmore preferably, not more than 20 amino acid substitutions. Of course,in order of ever-increasing preference, it is highly preferable for apolynucleotide which encodes the amino acid sequence of a TNF-gammapolypeptide to have an amino acid sequence which contains not more than10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions. Conservativesubstitutions are preferable.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production ofTNF-gamma polypeptides or peptides by recombinant techniques.

In accordance with a further aspect of the present invention, there isprovided a process for producing such polypeptide by recombinanttechniques comprising culturing recombinant prokaryotic and/oreukaryotic host cells, containing a human TNF-gamma nucleic acidsequence, under conditions promoting expression of said protein andsubsequent recovery of said protein.

The invention further provides an isolated TNF-gamma polypeptidecomprising an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of the full-length TNF-gamma-alphapolypeptide having the complete amino acid sequence shown in SEQ ID NO:2(i.e., positions −27 to 147 of SEQ ID NO:2); (b) the amino acid sequenceof the full-length TNF-gamma-alpha polypeptide having the complete aminoacid sequence shown in SEQ ID NO:2 excepting the N-terminal methionine(i.e., positions −26 to 147 of SEQ ID NO:2); (c) the amino acid sequenceof the predicted mature TNF-gamma-alpha polypeptide having the aminoacid sequence at positions 1-147 in SEQ ID NO:2; (d) the complete aminoacid sequence encoded by the cDNA clone HUVEO91 contained in the ATCCDeposit No. 75927; (e) the complete amino acid sequence excepting theN-terminal methionine encoded by the cDNA clone HUVEO91 contained in theATCC Deposit No. 75927; (f) the complete amino acid sequence of thepredicted mature TNF-gamma polypeptide encoded by the cDNA clone HUVEO91contained in the ATCC Deposit No. 75927; and (g) fragments of thepolypeptide of (a), (b), (c), (d), (e), or (f). The polypeptides of thepresent invention also include polypeptides having an amino acidsequence at least 80% identical, more preferably at least 90% identical,and still more preferably 95%, 96%, 97%, 98% or 99% identical to thosedescribed in (a), (b), (c), (d), (e) (f), or (g) above, as well aspolypeptides having an amino acid sequence with at least 90% similarity,and more preferably at least 95% similarity, to those above. Additionalembodiments of the invention relates to a polypeptide which comprisesthe amino acid sequence of an epitope-bearing portion of a TNF-gammapolypeptide having an amino acid sequence described in (a), (b), (c),(d), (e), (f), or (g) above. Peptides or polypeptides having the aminoacid sequence of an epitope-bearing portion of a TNF-gamma polypeptideof the invention include portions of such polypeptides with at least sixor seven, preferably at least nine, and more preferably at least about30 amino acids to about 50 amino acids, although epitope-bearingpolypeptides of any length up to and including the entire amino acidsequence of a polypeptide of the invention described above also areincluded in the invention.

The invention further provides an isolated TNF-gamma polypeptidecomprising an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of the full-length TNF-gamma-betapolypeptide having the complete amino acid sequence shown in SEQ IDNO:20 (i.e., positions 1 to 251 of SEQ ID NO:20); (b) the amino acidsequence of the full-length TNF-gamma-beta polypeptide having thecomplete amino acid sequence shown in SEQ ID NO:20 excepting theN-terminal methionine (i.e., positions 2 to 251 of SEQ ID NO:20); (c)the amino acid sequence of the predicted mature TNF-gamma-betapolypeptide having the amino acid sequence at positions 62-251 in SEQ IDNO:20; (d) the complete amino acid sequence encoded by the cDNA cloneHEMCZ56 contained in the ATCC Deposit No. 203055; (e) the complete aminoacid sequence excepting the N-terminal methionine encoded by the cDNAclone HEMCZ56 contained in the ATCC Deposit No. 203055; (f) the completeamino acid sequence of the predicted mature TNF-gamma polypeptideencoded by the cDNA clone contained in the ATCC Deposit No. 203055; and(g) fragments of the polypeptide of (a), (b), (c), (d), (e), or (f). Thepolypeptides of the present invention also include polypeptides havingan amino acid sequence at least 80% identical, more preferably at least90% identical, and still more preferably 95%, 96%, 97%, 98% or 99%identical to those described in (a), (b), (c), (d), (e) (f), or (g)above, as well as polypeptides having an amino acid sequence with atleast 90% similarity, and more preferably at least 95% similarity, tothose above. Additional embodiments of the invention relates to apolypeptide which comprises the amino acid sequence of anepitope-bearing portion of a TNF-gamma polypeptide having an amino acidsequence described in (a), (b), (c), (d), (e), (f), or (g) above.Peptides or polypeptides having the amino acid sequence of anepitope-bearing portion of a TNF-gamma polypeptide of the inventioninclude portions of such polypeptides with at least six or seven,preferably at least nine, and more preferably at least about 30 aminoacids to about 50 amino acids, although epitope-bearing polypeptides ofany length up to and including the entire amino acid sequence of apolypeptide of the invention described above also are included in theinvention.

A further embodiment of the invention relates to a polypeptide whichcomprises the amino acid sequence of a TNF-gamma polypeptide having anamino acid sequence which contains at least one amino acid substitution,but not more than 50 amino acid substitutions, even more preferably, notmore than 40 amino acid substitutions, still more preferably, not morethan 30 amino acid substitutions, and still even more preferably, notmore than 20 amino acid substitutions. Of course, in order ofever-increasing preference, it is highly preferable for a peptide orpolypeptide to have an amino acid sequence which comprises the aminoacid sequence of a TNF-gamma polypeptide, which contains at least one,but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acidsubstitutions. In specific embodiments, the number of additions,substitutions, and/or deletions in the amino acid sequence of FIGS.1A-C, FIGS. 20 A amd B, or fragments thereof (e.g., the extracellulardomain and/or other fragments described herein), is 1-5, 5-10, 5-25,5-50, 10-50 or 50-150, conservative amino acid substitutions arepreferable.

In another embodiment, the invention provides an isolated antibody thatbinds specifically to a TNF-gamma polypeptide having an amino acidsequence described above. The invention further provides methods forisolating antibodies that bind specifically to a TNF-gamma polypeptidehaving an amino acid sequence as described herein. Such antibodies areuseful diagnostically or therapeutically as described below.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing polynucleotides and/or polypeptidesof the invention to screen for agonists and antagonists, and fortherapeutic purposes, which include, but are not limited to, woundhealing, to inhibit tumor proliferation, to provide resistance toparasites, bacteria and viruses, to induce inflammatory activities, toinduce proliferation of endothelial cells and certain hematopoieticcells, to treat restenosis and to prevent certain autoimmune diseases.

In accordance with yet a further aspect of the present invention, thereare also provided nucleic acid probes comprising nucleic acid moleculesof sufficient length to specifically hybridize to human TNF-gammasequences.

In accordance with another aspect of the present invention, there areprovided TNF-gamma agonists which mimic TNF-gamma and binds to theTNF-gamma receptors to elicit TNF-gamma type responses.

In accordance with yet another aspect of the present invention, thereare provided antagonists to such polypeptides, which may be used toinhibit the action of such polypeptides, for example, to prevent septicshock, inflammation, cerebral malaria, activation of the HIV virus,graft rejection, bone resorption and cachexia.

In accordance with still another aspect of the present invention, thereare provided diagnostic assays for detecting diseases related to theunder-expression and over-expression of the TNF-gamma polypeptide andnucleic acid sequences encoding such polypeptide.

In a further aspect of the invention, TNF-gamma may be used to treatrheumatoid arthritis (RA) by inhibiting the increase in angiogensis orthe increase in endothelial cell proliferation required to sustain aninvading pannus in bone and cartilage as is often observed in RA.

In yet another aspect, the TNF-gamma may bind to a cell surface proteinwhich also functions as a viral receptor or coreceptor. Thus, TNF-gamma,or agonists or antagonists thereof, may be used to regulate viralinfectivity at the level of viral binding or interaction with theTNF-gamma receptor or coreceptor or during the process of viralinternalization or entry into the cell.

In accordance with all aspects of the invention, the term “TNF-gamma”refers to TNF-gamma-alpha and/or TNF-gamma-beta.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE FIGURES

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIGS. 1A-C illustrate the cDNA (SEQ ID NO:1) and corresponding deducedamino acid sequence (SEQ ID NO:2) of the polypeptide of TNF-gamma-alphaof the present invention. The initial 27 amino acids (underlined) arethe putative leader sequence. The standard one-letter abbreviations foramino acids are used. Potential asparagine-linked glycosylation sitesare marked in FIGS. 1A-C with a bolded asparagine symbol (N) in theTNF-gamma-alpha amino acid sequence and a bolded pound sign (#) abovethe first nucleotide encoding that asparagine residue in theTNF-gamma-alpha nucleolide sequence. Potential N-linked glycosylationsequences are found at the following locations in the TNF-gamma-alphaamino acid sequence: N-29 through N-32 (N-29, Y-30, T-31, N-32) andN-125 through D-128 (N-125, V-126, S-127, D128). Potential ProteinKinase C (PKC) phosphorylation sites are also marked in FIGS. 1A-C witha bolded threonine symbol (T) in the TNF-gamma-alpha amino acid sequenceand an asterisk (*) above the first nucleotide encoding that threonineresidue in the TNF-gamma-alpha nucleotide sequence. Potential PKCphosphorylation sequences are found at the following locations in theTNF-gamma-alpha amino acid sequence: T-32 through K-34 (T-32, N-33,K-34) and T-50 through R-52 (T-50, F-51, R-52). Potential Casein KinaseII (CK2) phosphorylation sites are also marked in FIGS. 1A and 1B with abolded serine or threonine symbol (S or T) in the TNF-gamma-alpha aminoacid sequence and an asterisk (*) above the first nucleotide encodingthe appropriate serine or threonine residue in the TNF-gamma-alphanucleotide sequence. Potential CK2 phosphorylation sequences are foundat the following locations in the TNF-gamma-alpha amino acid sequence:S-83 through E-86 (S-83, Y-84, P-85, E-86); S-96 through E-99 (S-96,V-97, C-98, E-99); S-115 through E-118 (S-115, L-116, Q-117, E-118);S-130 through D-133 (S-130, L-131, V-132, D-133); and T-135 throughD-138 (T-135, K-136, E-137, D-138). Potential myristylation sites arealso marked in FIGS. 1A-C with a double underline in the TNF-gamma-alphaamino acid sequence. Potential myristylation sequences are found at thefollowing locations in the TNF-gamma-alpha amino acid sequence: G-20through K-25 (G-20, L-21, A-22, F-23, T-24, K-25) and G-111 throughL-116 (G-111, A-112, M-113, F-114, S-115, L-116).

FIGS. 2A-C illustrate an amino acid sequence alignment betweenTNF-gamma-alpha (SEQ ID NO:2) and other members of the TNF familyincluding human TNF-alpha (GenBank No. Z15026; SEQ ID NO:3), humanTNF-beta (GenBank No. Z15026; SEQ ID NO:4), human lymphotoxin-beta(LTbeta; GenBank No. L11016; SEQ ID NO:5), and rat Fas Ligand (FASL:GenBank No. U03470, SEQ ID NO:6). TNF-gamma contains the conserved aminoacid residues of the TNF family as shown by the boxed and shaded areas.The aligned molecules are presented in their entirety as FIGS. 2A, 2B,and 2C.

FIG. 3A is an RNA blot analysis showing the human tissues whereTNF-gamma is expressed. RNA from the tissues indicated were probed withlabeled TNF-gamma cDNA. TNF-gamma-alpha mRNA exists predominantly in thekidney since FIG. 3A shows a distinct band. Other lanes seem to showstrong hybridization, however, these are actually non-specific smears.

FIG. 3B is an RNA blot analysis showing that TNF-gamma is expressedpredominantly in HUVEC cells (human umbilical vein endothelial cells;lane 9). Lane 6 and lane 8 are non-specific smears. RNA from the celllines indicated were probed with labeled TNF-gamma-alpha cDNA. Lane 1 isCAMA1 (breast cancer); lane 2 AN3CA (uterine cancer); lane 3, SK.UT.1(uterine cancer); lane 4, MG63 (osteoblastoma); lane 5, HOS(osteoblastoma); lane 6, MCF7 (breast cancer); lane 7, OVCAR-3 (ovariancancer); lane 8, CAOV-3 (ovarian cancer); lane 9, HUVEC; lane 10, AOSMIC(smooth muscle); lane 11, foreskin fibroblast.

FIG. 4 illustrates the relative expression of TNF-gamma in proliferatingor quiescent endothelial cells. The TNF-gamma mRNA levels in culturedHUVEC cells were determined by Northern blotting analysis. Identicalamounts of total RNA (15 μg) were loaded on each lane, as indicated bythe intensity of beta-actin. The signal which corresponds to TNF-gammais designated “VEGI”. Total RNA was prepared at the indicated time point(days post-seeding). The number of cells in each culture to flask wasdetermined simultaneously. The experiment was carried out in duplicate.Cells were seeded at 125,00 cells per flask (T-25).

FIG. 5 is a photograph of a polyacrylamide gel electrophoresis analysisof TNF-gamma protein. TNF-gamma was produced by bacterial expression andpurified as described in Example 1.

FIG. 6 is a photograph of a gel showing the relative purity and mobilityof baculovirus-expressed TNF-gamma. The expression and purification ofTNF-gamma using the baculovirus system is described in Example 2.

FIG. 7A consists of photographs of WEHI 164 cells which are untreated(FIG. 7Aa) and after exposure to TNF-alpha (FIG. 7Ab), TNF-gamma (FIG.7Ac), and TNF-beta (FIG. 7Ad). Cells which have an elongated non-roundmorphology have been lysed. The various TNF molecules were added at aconcentration of approximately 0.5 μg/ml. Photographs were taken 72hours after addition of the various TNF molecules.

FIG. 7B illustrates the ability of TNF-gamma (FIG. 7Bc) in comparison toTNF-alpha (FIG. 7Ba) and TNF-beta (FIG. 7Bb) to inhibit WEHI 164 cellgrowth.

FIG. 8 illustrates the ability of recombinant TNF-alpha (FIG. 8B),TNF-beta (FIG. 8D), and TNF-gamma (FIG. 8C) to induce morphologicalchange in L929 cells with respect to untreated L929 cells (FIG. 8A). Themorphology change is indicated by dark round cells. Cells were treatedwith the various recombinant TNF molecules (produced in E. coli) atapproximately 0.5 μg/ml. The photographs were taken 72 hours after theaddition of the various TNF molecules. The morphology change indicatesthat the cells have been killed.

FIG. 9 is a graphical illustration of the effect of TNF-gamma (FIG. 9C),TNF-alpha (FIG. 9A), and TNF-beta (FIG. 9B) on venous endothelial cells.Cell proliferation after endothelial cells were treated withcommercially available TNF-alpha and TNF-beta and E. coli producedTNF-gamma was quantified using an MTS assay.

FIG. 10 shows the effect of TNF-gamma on the proliferation ofendothelial cell and breast cancer cells. The number of cells areplotted against TNF-gamma concentration as indicated (TNF-gamma isdesignated “VEGI” in this figure). Inhibition of the growth of adultbovine aortic endothelial (ABAE) cells (dark circles), but not that ofMDA-MB-231 (dark triangles) or MDA-MB-435 (open circles) cells, isshown. The cells were seeded at 2×10³ cells/well in triplicate in24-well plates. The ABAE cell culture media contained IMEM (LifeTechnologies, Inc., Rockville, Md.) supplemented with 10% FCS and (1ng/ml) FGF-2. The cultures were maintained at 37° C., 5% CO₂, for 6days. The cells were then trypsinized, and the number of cellsdetermined by using a Coulter counter. One-fifth of the total number ofrecovered ABAE cells is shown in order to normalize the comparison withthe MDA-MB-231 and MDA-MB-435 cells.

FIG. 11 is a photograph of HL 60 cells with control (FIG. 11A) showingthe HL60 cells being spread apart; TNF-alpha (FIG. 11B) and TNF-gamma(FIG. 11C) induce cell adhesion and cell-cell contact as illustrated bythe cells adhering together in the lower right.

FIG. 12 illustrates the ability of recombinant TNF-gamma (represented bysquares), TNF-alpha (represented by circles) and TNF-beta (representedby triangles) to induce WEHI 164 cell death. Cell death is inverselyproportional to the ratio of absorbance at 405 nm to that at 490 nm).

FIG. 13 illustrates that TNF-gamma does not significantly bind to twoknown soluble TNF receptors, namely sTNF RI (p55; solid bars) and sTNFRII (p75; hatched bars).

FIG. 14 demonstrates the effect of TNF-gamma on the ability of ABAEcells to form capillary-like tubes on collagen gels. The ability ofrecombinant TNF-gamma (residues 12-147 as shown in SEQ ID NO:2 anddesignated “VEGI” in this figure) to inhibit the formation ofcapillary-like tubes by ABAE cells is shown. The p-values (t-test) givenabove the columns are obtained by comparing the extent of thecapillary-like tube formation by ABAE cells in the presence of variousconcentrations of TNF-gamma, as indicated, to that when TNF-gamma isabsent from the culture media.

FIG. 15 shows the inhibition of angiogenesis in collagen gels placed onchicken embryonic chorioallantoic membrane (CAM) by TNF-gamma. Thegrowth of new capillary vessels into collagen gel pellets placed on theCAM was induced by either FGF-2 (100 ng) or VEGF (250 ng). The extent ofangiogenesis in the gels was determined by evaluation of thefluorescence intensity of FITC-dextran injected into the CAMcirculation. Inhibition of the capillary vessel growth by therecombinant TNF-gamma (designated “VEGI” in this figure), as indicatedby a lower value than 100, is shown. The experiment was carried out intriplicate.

FIG. 16 illustrates the inhibition of growth of human breast cancerxenograft tumors in athymic nude mice by TNF-gamma. Mixtures ofTNF-gamma-overexpressing or vector-transfected CHO cells (5×10⁶ cellsper injection) and human breast cancer cells (1×10⁶ cells per injection)were injected into the mammary fat pads of the nude mice. Tumor sizes(area) were monitored following injection. The sizes of the MDA-MB-231xenograft tumors (mm²) were plotted as a function of dayspost-inoculation (FIG. 16A). The sizes of the MDA-MB-435 xenografttumors (mm²) were plotted as a function of days post-inoculation (FIG.16B). Open circles represent values of tumors co-inoculated withvector-transfected CHO cells, whereas closed circles represent values oftumors co-inoculated with TNF-gamma-transfected CHO cells.

FIG. 17 shows an analysis of the TNF-gamma-alpha amino acid sequence(SEQ ID NO:2). Alpha, beta, turn and coil regions; hydrophilicity andhydrophobicity; amphipathic regions; flexible regions; antigenic indexand surface probability are shown, as predicted using the defaultparameters of the recited computer programs. In the “Antigenic Index orJameson-Wolf” graph, the positive peaks indicate locations of the highlyantigenic regions of the TNF-gamma protein, i.e., regions from whichepitope-bearing peptides of the invention can be obtained.

FIGS. 18A-D show an alignment of the nucleotide sequences ofTNF-gamma-alpha (SEQ ID NO:1) and TNF-gamma-beta (SEQ ID NO:19)constructed by using the computer program BESTFIT set at defaultparameters.

FIG. 19 shows an alignment of the amino acid sequences ofTNF-gamma-alpha (SEQ ID NO:2) and TNF-gamma-beta (SEQ ID NO:20)constructed using the default parameters of the computer programBESTFIT.

FIGS. 20A and B illustrate the cDNA (SEQ ID NO:19) and correspondingdeduced amino acid sequence (SEQ ID NO:20) of the polypeptide of theTNF-gamma-beta of the present invention. The standard one-letterabbreviations for amino acids are used. Amino acids methionine-1 totryptophan-35 are the predicted intracellular domain. Amino acidresidues alanine-36 through alanine-61 (underlined) are the putativetransmembrane sequence. Amino acid residues glutamine-62 throughleucine-251 (underlined) are the putative transmembrane sequence.Potential asparagine-linked glycosylation sites are marked in FIGS. 20Aand B with a bolded asparagine symbol (N) in the TNF-gamma-beta aminoacid sequence and a bolded pound sign (#) above the first nucleotideencoding that asparagine residue in the TNF-gamma-alpha nucleotidesequence. Potential N-linked glycosylation sequences are found at thefollowing locations in the TNF-gamma-beta amino acid sequence: N-133through N-136 (N-133, Y-134, T-135, N-136) and N-229 through D-232(N-229, V-230, S-231, D-232). Potential Protein Kinase C (PKC)phosphorylation sites are also marked in FIGS. 20A and B with a boldedserine or threonine symbol (S or T) in the TNF-gamma-beta amino acidsequence and an asterisk (*) above the first nucleotide encoding thatthreonine residue in the TNF-gamma-beta nucleotide sequence. PotentialPKC phosphorylation sequences are found at the following locations inthe TNF-gamma-beta amino acid sequence: S-23 through R-25 (S-23, C-24,R-25); S-32 through R-34 (S-32, A-33, R-34); T-135 through K-137 (T-135,N-136, K-137); and T-154 through R-156 (T-154, F-155, R-156). PotentialCasein Kinase II (CK2) phosphorylation sites are also marked in FIGS.20A and B with a bolded serine or threonine symbol (S or T) in theTNF-gamma-beta amino acid sequence and an asterisk (*) above the firstnucleotide encoding the appropriate serine or threonine residue in theTNF-gamma-beta nucleotide sequence. Potential CK2 phosphorylationsequences are found at the following locations in the TNF-gamma-betaamino acid sequence: S-8 through E-11 (S-8, F-9, G-10, E-11); S-187through E-190 (S-187, Y-188, P-189, E-190); S-200 through E-203 (S-200,V-201, C-202, E-203); S-219 through E-222 (S-219, L-220, Q-221, E-222);S-234 through D237 (S-234, L-235, V-236, D-237); and T-239 through D-242(T-239, K-240, E-241, D-242). Potential myristylation sites are alsomarked in FIGS. 20A and B with a double underline in the TNF-gamma-betaamino acid sequence. Potential myristylation sequences are found at thefollowing locations in the TNF-gamma-beta amino acid sequence: G-6through E-11 (G-6, L-7, S-8, F-9, G-10, E-11); G-124 through G-129(G-124, L-125, A-126, F-127, T-128, K-129); and G-215 through L-220(G-215, A-216, M-217, F-218, S-219, L-220).

DETAILED DESCRIPTION

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding a TNF-gamma-alpha polypeptidehaving the amino acid sequence shown in FIGS. 1A-C (SEQ ID NO:2), whichwas determined by sequencing a cloned cDNA (HUVE091). As shown in FIGS.2A-2C, the TNF-gamma-alpha polypeptide of the present invention sharessequence homology with human TNF-alpha (SEQ ID NO:3), TNF-beta (SEQ IDNO:4), human lymphotoxin-beta (SEQ ID NO:5) and FAS ligand (SEQ IDNO:6). The TNF-gamma-alpha of the invention functions at least in theinhibition of angiogenesis, as an anti-tumor cytokine-like molecule, asa treatment for arthritis by the inhibition of angiogenesis and/orendothelial cell proliferation associated with invading pannus in boneand cartilage, as an inducer of NF-kB and c-Jun kinase (JNK), an inducerof cell adhesion, and as an inducer apoptosis (See Examples,particularly Examples 12-15). The nucleotide sequence shown in SEQ IDNO:1 was obtained by sequencing a cDNA clone (HUVE091), which wasdeposited on Oct. 26, 1994 at the American Type Culture Collection,10801 University Boulevard, Mannasas, Va. 20110-2209, and givenaccession number 75927. The deposited plasmid is contained inpBluescript SK(−) plasmid (Stratagene, La Jolla, Ca.). Furthercharacterization of the protein encoded by clone HUVE091 is presented incopending U.S. Provisional Application Ser. No. 60/074,047, filed Feb.9, 1998; the entire disclosure of which is hereby incorporated byreference.

The present invention also provides isolated nucleic acid moleculescomprising a polynucleotide (SEQ ID NO:19) encoding a TNF-gamma-betapolypeptide having the amino acid sequence shown in FIGS. 20A and B (SEQID NO:20), which was determined by sequencing a cloned cDNA (HEMCZ56).The TNF-gamma-beta polypeptide is a splice variant of theTNF-gamma-alpha polypeptide disclosed herein. The nucleotide sequenceshown in SEQ ID NO:19 was obtained by sequencing a cDNA clone (HEMCZ56),which was deposited on Jul. 9, 1998 at the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209, andgiven accession number 203055. The deposited plasmid is contained inpBluescript SK(−) plasmid (Stratagene, La Jolla, Ca.).

Nucleic Acid Molecules

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc., FosterCity, Ca.), and all amino acid sequences of polypeptides encoded by DNAmolecules determined herein were predicted by translation of a DNAsequence determined as above. Therefore, as is known in the art for anyDNA sequence determined by this automated approach, any nucleotidesequence determined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art. As is also known inthe art, a single insertion or deletion in a determined nucleotidesequence compared to the actual sequence will cause a frame shift intranslation of the nucleotide sequence such that the predicted aminoacid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

By “nucleotide sequence” of a nucleic acid molecule or polynucleotide isintended, for a DNA molecule or polynucleotide, a sequence ofdeoxyribonucleotides, and for an RNA molecule or polynucleotide, thecorresponding sequence of ribonucleotides (A, G, C and U), where eachthymidine deoxyribonucleotide (T) in the specified deoxyribonucleotidesequence is replaced by the ribonucleotide uridine (U).

Using the information provided herein, such as the nucleotide sequencein FIGS. 1A-C (SEQ ID NO:1), or the nucleotide sequence in FIGS. 20A andB (SEQ ID NO:19) a nucleic acid molecule (i.e., polynucleotide) of thepresent invention encoding a TNF-gamma-alpha or TNF-gamma-betapolypeptide may be obtained using standard cloning and screeningprocedures, such as, for example, those for cloning cDNAs using mRNA asthe starting material. For example, polynucleotides encodingTNF-gamma-alpha polypeptides may routinely be obtained from any cell ortissue source that expresses TNF-gamma-alpha, such as, for example,human kidney and umbilical vein endothelial cells. Illustrative of theinvention, the nucleic acid molecules described in FIGS. 1A-C (SEQ IDNO:1) was discovered in a cDNA library derived from human umbilical veinendothelial cells. The cDNA clone corresponding to TNF-gamma-alpha(clone HUVE091) contains an open reading frame encoding a protein of 174amino acid residues of which approximately the first 27 amino acidsresidues are the putative leader sequence such that the mature proteincomprises 147 amino acids. The protein exhibits the highest degree ofhomology at the C-terminus to Rabbit TNF-alpha (Ito, H., et al., DNA5:157-165 (1986); GenBank Accession No. M12846; SEQ ID NO:7) with 38%identity and 58% similarity over a 111 amino acid stretch. Sequencesconserved throughout the members of the TNF family are also conserved inTNF-gamma (see FIGS. 2A-2C). The shaded letters indicate conserved aminoacid residues. The TNF-gamma mRNA is specifically expressed in humanumbilical vein endothelial cells as shown in the RNA blot analysis ofFIG. 3B.

Further, polynucleotides encoding a TNF-gamma-beta polypeptide mayroutinely be obtained from induced and resting endothelial cells,umbilical vein, tonsils, and several other cell and tissue types.Illustrative of the invention, the nucleic acid molecules described inFIGS. 20A and B (SEQ ID NO:19) was discovered in a cDNA library derivedfrom induced endothelial cells. The cDNA clone corresponding toTNF-gamma-beta (HEMCZ56) contains an open reading frame encoding aprotein of 251 amino acid residues of which approximately the first 35amino acids residues are the putative intracellular domain and aminoacids 36-61 are a putative transmembrane domain and amino acid residues62-251 are a putative extracellular domain.

The amino acid residues constituting the extracellular, transmembrane,and intracellular domains have been predicted by computer analysis.Thus, as one of ordinary skill would appreciated, the amino acidresidues constituting these domains may vary slightly (e.g., by about 1to about 15 amino acid residues) depending on the criteria used todefine each domain.

In accordance with an aspect of the present invention, there is providedan isolated nucleic acid (polynucleotide) which encodes for the maturepolypeptide having the deduced amino acid sequence of FIGS. 1A-C (SEQ IDNO:2), or for the mature polypeptide encoded by the cDNA of the clonedesignated HUVEO91 deposited as ATCC Deposit No. 75927 on Oct. 26, 1994.

In addition, in accordance with another aspect of the present invention,there is provided an isolated nucleic acid (polynucleotide) whichencodes for the mature polypeptide having the deduced amino acidsequence of FIGS. 20A and B (SEQ ID NO:20), or for the maturepolypeptide encoded by the cDNA of the clone designated HEMCZ56deposited as ATCC Deposit No. 203055 on Jul. 9, 1998.

By “isolated” nucleic acid molecule(s) or polynucleotide is intended amolecule, DNA or RNA, which has been removed form its nativeenvironment. For example, recombinant DNA molecules (polynucleotides)contained in a vector are considered isolated for the purposes of thepresent invention. Further examples of isolated DNA molecules(polynucleotides) include recombinant DNA molecules maintained inheterologous host cells or purified (partially or substantially) DNAmolecules in solution. Isolated RNA molecules (polynucleotides) includein vivo or in vitro RNA transcripts of the DNA molecules(polynucleotides) of the present invention. Isolated nucleic acidmolecules or polynucleotides according to the present invention furtherinclude such molecules produced synthetically.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand.

Isolated nucleic acid molecules of the present invention include thepolynucleotide sequence depicted in FIGS. 1A-C (SEQ ID NO:1) encodingthe mature TNF-gamma-alpha polypeptide, the polynucleotide sequencedepicted in FIGS. 20A and B (SEQ ID NO:19) encoding the matureTNF-gamma-beta polypeptide, the polynucleotide sequences contained indeposited clone (HUVE091) deposited as ATCC Deposit No. 75927 encodingthe mature TNF-gamma-alpha polypeptide, the polynucleotide sequencescontained in deposited clone (HEMCZ56) deposited as ATCC Deposit No.203055 encoding the mature TNF-gamma-beta polypeptide, andpolynucleotide sequences which comprise a sequence different from thosedescribed above, but which due to the degeneracy of the genetic code,encodes the same mature polypeptide as the DNA of FIGS. 1A and B, FIGS.20A and B, or the deposited cDNA. Of course, the genetic code is wellknown in the art. Thus, it would be routine for one skilled in the artto generate such degenerate variants.

The amino acid sequence of the complete TNF-gamma-alpha protein includesa leader sequence and a mature protein, as shown in FIGS. 1A-C (SEQ IDNO:2). More in particular, the present invention provides nucleic acidmolecules encoding a mature form of the TNF-gamma-alpha protein. Thus,according to the signal hypothesis, once export of the growing proteinchain across the rough endoplasmic reticulum has been initiated,proteins secreted by mammalian cells have a signal or secretory leadersequence which is cleaved from the complete polypeptide to produce asecreted “mature” form of the protein. Most mammalian cells and eveninsect cells cleave secreted proteins with the same specificity.However, in some cases, cleavage of a secreted protein is not entirelyuniform, which results in two or more mature species of the protein.Further, it has long been known that the cleavage specificity of asecreted protein is ultimately determined by the primary structure ofthe complete protein, that is, it is inherent in the amino acid sequenceof the polypeptide. Therefore, the present invention provides anucleotide sequence encoding the mature TNF-gamma-alpha polypeptidehaving the amino acid sequence encoded by the cDNA clone contained inATCC Deposit No. 75927. By the “mature TNF-gamma-alpha polypeptidehaving the amino acid sequence encoded by the cDNA clone in ATCC DepositNo. 75927” is meant the mature form(s) of the TNF-gamma-alpha proteinproduced by expression in a mammalian cell (e.g., COS cells, asdescribed below) of the complete open reading frame encoded by the humanDNA sequence of the deposited clone.

The polynucleotide which encodes for the mature polypeptide of FIGS. 20Aand B or for the mature polypeptide encoded by the deposited cDNA(HEMCZ56) may include, but is not limited to: only the coding sequencefor the mature polypeptide, the coding sequence for the maturepolypeptide and additional coding sequence such as a leader or secretorysequence or a transmembrane sequence or a proprotein sequence; thecoding sequence for the mature polypeptide (and optionally additionalcoding sequence) and non-coding sequence, such as introns or non-codingsequence 5′ and/or 3′ of the coding sequence for the mature polypeptide.

The present invention also includes polynucleotides, wherein the codingsequence for the mature polypeptide may be fused in the same readingframe to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5′ amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains.

Thus, for example, the polynucleotide of the present invention mayencode for a mature protein, or for a protein having a prosequence orfor a protein having both a prosequence and a presequence (leadersequence).

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

Thus, the term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequence.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments (i.e., portions),analogs and derivatives of the polypeptide having the deduced amino acidsequence of FIGS. 1A-C, FIGS. 20A and B, and the polypeptide encoded bythe cDNA of the deposited clones. The variant of the polynucleotide maybe a naturally occurring allelic variant of the polynucleotide or anon-naturally occurring variant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the samemature polypeptide as shown in FIGS. 1A-C, or the mature polypeptideencoded by the cDNA of the deposited clone HUVEO91 as well as variantsof such polynucleotides which variants encode for a fragment, derivativeor analog of the polypeptide of FIGS. 1A-C, or the polypeptide encodedby the cDNA of the deposited clone HUVEO91. Such nucleotide variantsinclude deletion variants, substitution variants and addition orinsertion variants.

Additionally, the present invention includes polynucleotides encodingthe mature polypeptide as shown in FIGS. 20A and B, or the maturepolypeptide encoded by the cDNA of the deposited clone HEMCZ56 as wellas variants of such polynucleotides which variants encode for afragment, derivative or analog of the polypeptide of FIGS. 20A and B, orthe polypeptide encoded by the cDNA of the deposited clone HEMCZ56. Suchnucleotide variants include deletion variants, substitution variants andaddition or insertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIGS. 1A-C or of the coding sequence of the deposited cloneHUVEO91. Alternatively, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIGS. 20A and B or of the coding sequence of the depositedclone HEMCZ56. As known in the art, an allelic variant is an alternateform of a polynucleotide sequence which may have a substitution,deletion or addition of one or more nucleotides, which does notsubstantially alter the function of the encoded polypeptide.

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatednucleic acid molecule having the nucleotide sequence of the depositedcDNAs (HUVEO91 and HEMCZ56), or the nucleotide sequence shown in FIGS.1A-C (SEQ ID NO:1), FIGS. 20A and B (SEQ ID NO:19), or the complementarystrand thereto, is intended fragments at least 15 nt, and morepreferably at least 20 nt, still more preferably at least 30 nt, andeven more preferably, at least 40, 50, 100, 150, 200, 250, 300, 400, or500 nt in length. These fragments have numerous uses which include, butare not limited to, diagnostic probes and primers as discussed herein.Of course, larger fragments 50-1500 nt in length are also usefulaccording to the present invention as are fragments corresponding tomost, if not all, of the nucleotide sequence of the deposited cDNA cloneHUVEO91, the deposited cDNA clone HEMCZ56, the nucleotide sequencedepicted in FIGS. 1A-C (SEQ ID NO:1), or the nucleotide sequencedepicted in FIGS. 20A and B (SEQ ID NO 20). By a fragment at least 20 ntin length, for example, is intended fragments which include 20 or morecontiguous bases from the nucleotide sequence of the deposited cDNAclones (HUVEO91 and HEMCZ56), the nucleotide sequence as shown in FIGS.1A-C (SEQ ID NO:1), or the nucleotide sequence as shown in FIGS. 20A andB.

In specific embodiments, the polynucleotide fragments of the inventionencode a polypeptide which demonstrates a functional activity. By apolypeptide demonstrating “functional activity” is meant, a polypeptidecapable of displaying one or more known functional activities associatedwith a complete or mature TNF-gamma polypeptide. Such functionalactivities include, but are not limited to, biological activity ((e.g.,inhibition of angiogenesis, inhibition of endothelial cellproliferation, induction of NF-κB and c-Jun kinase (JNK), induction ofcell adhesion, and induction of apoptosis (See Examples, particularlyExamples 12-15)), antigenicity [ability to bind (or compete with aTNF-gamma polypeptide for binding) to an anti-TNF-gamma antibody],immunogenicity (ability to generate antibody which binds to a TNF-gammapolypeptide), the ability to form polymers with other TNF-gammapolypeptides, and ability to bind to a receptor or ligand for aTNF-gamma polypeptide (e.g. DR3).

The invention also provides nucleic acid molecules having nucleotidesequences related to extensive fragments of SEQ ID NO:1 which have beendetermined from the following related cDNA clones: HUVEO91 (SEQ IDNO:8), HMPAP05 (SEQ ID NO:9), HSXCA44 (SEQ ID NO:10), HEMFG66 (SEQ IDNO:11), and HELAM93 (SEQ ID NO:12).

The invention also provides nucleic acid molecules having nucleotidesequences related to extensive fragments of SEQ ID NO:19 which have beendetermined from the following related cDNA clones: HUVEO91P01 (SEQ IDNO:21), HMPTI24R (SEQ ID NO:22), HELAM93R (SEQ ID NO:23), and HEMFG66R(SEQ ID NO:24).

In specific embodiments, the polynucleotide fragments of the inventioncomprise, or alternatively, consist of, a polynucleotide comprising anyportion of at least 30 nucleotides, preferably at least 50 nucleotides,of SEQ ID NO:1 from nucleotide residue 1 to 2442, preferably excludingthe nucleotide sequences determined from the abovelisted cDNA clones.Representative examples of the TNF-gamma-alpha polynucleotide fragmentsof the invention, include fragments that comprise, or alternatively,consist of, nucleotides: 783-1304, 800-1300, 850-1300, 900-1300,950-1300, 1000-1300, 1050-1300, 1100-1300, 1150-1300, 1200-1300,1250-1300, 783-1250, 800-1250, 850-1250, 900-1250, 950-1250, 1000-1250,1050-1250, 1100-1250, 1150-1250, 1200-1250, 783-1200, 800-1200,850-1200, 900-1200, 950-1200, 1000-1200, 1050-1200, 1100-1200,1150-1200, 783-1150, 800-1150, 850-1150, 900-1150, 950-1150, 1000-1150,1050-1150, 1100-1150, 783-1100, 800-1100, 850-1100, 900-1100, 950-1100,1000-1100, 1050-1100, 783-1050, 800-1050, 850-1050, 900-1050, 950-1050,1000-1050, 783-1000, 800-1000, 850-1000, 900-1000, 950-1000, 783-950,800-950, 850-950, 900-950, 783-900, 800-900, and 850-900 of SEQ ID NO:1,or the complementary polynucleotide strand thereto, or the cDNAcontained in the deposited clone HUVEO91.

In additional specific embodiments, the polynucleotide fragments of theinvention comprise, or alternatively, consist of, a polynucleotidecomprising any portion of at least 30 nucleotides, preferably at least50 nucleotides, of SEQ ID NO:19 from nucleotide residue 1 to 1116,preferably excluding the nucleotide sequences determined from theabovelisted cDNA clones (i.e., list from p.25).

Preferred embodiments of the invention encompass polynucleotidesencoding polypeptides comprising, or alternatively consisting of, theamino acid sequence of residues −1-147 (i.e., −1 to 147), 1-147 (i.e.,+1 to 147), 2-147, 3-147, 4-147, 5-147, 6-147, 7-147, 8-147, 9-147,10-147, 11-147, 12-147, and 13-147 of SEQ ID NO:2. Polynucleotidesencoding these polypeptides are also provided.

Representative examples of the TNF-gamma-beta polynucleotide fragmentsof the invention, include fragments that comprise, or alternatively,consist of, nucleotides 1-1116, 50-1116, 100-1116, 150-1116, 200-1116,250-1116, 300-1116, 350-1116, 400-1116, 450-1116, 500-1116, 550-1116,600-1116, 650-1116, 700-1116, 750-1116, 800-1116, 850-1116, 900-1116,950-1116, 1000-1116, 1050-1116, 1-1100, 50-1100, 100-1100, 150-1100,200-1100, 250-1100, 300-1100, 350-1100, 400-1100, 450-1100, 500-1100,550-1100, 600-1100, 650-1100, 700-1100, 750-1100, 800-1100, 850-1100,900-1100, 950-1100, 1000-1100, 1050-1100, 1-1050, 50-1050, 100-1050,150-1050, 200-1050, 250-1050, 300-1050, 350-1050, 400-1050, 450-1050,500-1050, 550-1050, 600-1050, 650-1050, 700-1050, 750-1050, 800-1050,850-1050, 900-1050, 950-1050, 1000-1050, 1-1000, 50-1000, 100-1000,150-1000, 200-1000, 250-1000, 300-1000, 350-1000, 400-1000, 450-1000,500-1000, 550-1000, 600-1000, 650-1000, 700-1000, 750-1000, 800-1000,850-1000, 900-1000, 950-1000, 1-950, 50-950, 100-950, 150-950, 200-950,250-950, 300-950, 350-950, 400-950, 450-950, 500-950, 550-950, 600-950,650-950, 700-950, 750-950, 800-950, 850-950, 900-950, 1-900, 50-900,100-900, 150-900, 200-900, 250-900, 300-900, 350-900, 400-900, 450-900,500-900, 550-900, 600-900, 650-900, 700-900, 750-900, 800-900, 850-900,1-850, 50-850, 100-850, 150-850, 200-850, 250-850, 300-850, 350-850,400-850, 450-850, 500-850, 550-850, 600-850, 650-850, 700-850, 750-850,800-850, 1-800, 50-800, 100-800, 150-800, 200-800, 250-800, 300-800,350-800, 400-800, 450-800, 500-800, 550-800, 600-800, 650-800, 700-800,750-800, 1-750, 50-750, 100-750, 150-750, 200-750, 250-750, 300-750,350-750, 400-750, 450-750, 500-750, 550-750, 600-750, 650-750, 700-750,1-700, 50-700, 100-700, 150-700, 200-700, 250-700, 300-700, 350-700,400-700, 450-700, 500-700, 550-700, 600-700, 650-700, 1-650, 50-650,100-650, 150-650, 200-650, 250-650, 300-650, 350-650, 400-650, 450-650,500-650, 550-650, 600-650, 1-600, 50-600, 100-600, 150-600, 200-600,250-600, 300-600, 350-600, 400-600, 450-600, 500-600, 550-600, 1-550,50-550, 100-550, 150-550, 200-550, 250-550, 300-550, 350-550, 400-550,450-550, 500-550, 1-500, 50-500, 100-500, 150-500, 200-500, 250-500,300-500, 350-500, 400-500, 450-500, 1-450, 50-450, 100450, 150-450,200-450, 250-450, 300-450, 350-450, 400-450, 1-400, 50-400, 100-400,150-400, 200-400, 250-400, 300-400, 350-400, 1-350, 50-350, 100-350,150-350, 200-350, 250-350, 300-350, 1-300, 50-300, 100-300, 150-300,200-300, 250-300, 1-250, 50-250, 100-250, 150-250, 200-250, 1-200,50-200, 100-200, 150-200, 1-150, 50-150, 100-150, 1-100, 50-100, and1-50 of SEQ ID NO:19 or the complementary polynucleotide strand thereto,or the cDNA contained in the deposited clone HEMCZ56.

Preferred nucleic acid fragments of the present invention also includenucleic acid molecules encoding one or more of the following domains ofTNF-gamma-alpha (e.g., as described also in the legend to FIGS. 1A-C):potential asparagine-linked glycosylation sites N-29 through N-32 (N-29,Y-30, T-31, N-32) and N-125 through D-128 (N-125, V-126, S-127, D-128);potential Protein Kinase C (PKC) phosphorylation sites T-32 through K-34(T-32, N-33, K-34) and T-50 through R-52 (T-50, F-51, R-52); potentialCasein Kinase II (CK2) phosphorylation sites S-83 through E-86 (S-83,Y-84, P-85, E-86); S-96 through E-99 (S-96, V-97, C-98, E-99); S-115through E-118 (S-115, L-116, Q-117, E-118); S-130 through D-133 (S-130,L-131, V-132, D133); and T-135 through D-138 (T-135, K-136, E-137,D-138); and potential myristylation sites G-20 through K-25 (G-20, L-21,A-22, F-23, T24, K-25) and G-111 through L-116 (G-111, A-112, M-113,F-114, S-115, L-116) of SEQ ID NO:2.

Among the especially preferred polynucleotides of the invention arethose characterized by encoding structural or functional attributes ofTNF-gamma. Such polynucleotides encode amino acid residues that comprisealpha-helix and alpha-helix forming regions (“alpha-regions”),beta-sheet and beta-sheet forming regions (“beta regions”), turn andturn-forming regions (“turn-regions”), coil and coil-forming regions(“coil regions”), hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, surface forming regions,and high antigenic index regions (i.e., having an antigenic regions ofthree or more contiguous amino acid residues each of which having anantigenic index of greater than or equal to 1.5) of TNF-gamma. Certainpreferred regions are those set out in FIG. 17, and include, but are notlimited to, regions of the aforementioned types identified by analysisof the amino acid sequence depicted in FIG. 1 (SEQ ID NO:2) using thedefault parameters of the identified computer programs, such preferredregions include; Garnier-Robson alpha-regions, beta-regions,turn-regions, and coil-regions, Chou-Fasman alpha-regions beta-regions,and coil-regions, Kyte-Doolittle hydrophilic regions and hydrophobicregions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulzflexible regions, Emini surface-forming regions and Jameson-Wolf regionsof high antigenic index.

Data which represent TNF-gamma-beta in a fashion as described above forTNF-gamma-alpha (see FIG. 17) may easily be prepared using the aminoacid sequence shown in FIGS. 20A and 20B and in SEQ ID NO:20. As such,each of the abovelisted structural or functional attributes of TNF-gammalisted above (i.e. Garnier-Robson alpha-regions, beta-regions,turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions,and coil-regions, Kyte-Doolittle hydrophilic regions and hydrophobicregions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulzflexible regions, Emini surface-forming regions and Jameson-Wolf regionsof high antigenic index, etc.) apply equally well to TNF-gamma-alpha andTNF-gamma-beta.

Certain preferred regions in these regards are set out in FIG. 17, butmay also be represented or identified by using a tabular representationof the data presented in FIG. 17. The DNA*STAR computer algorithm usedto generate FIG. 17 (set on the original default parameters) will easilypresent the data in FIG. 17 in such a tabular format. A tabular formatof the data in FIG. 17 may be used to easily determine specificboundaries of a preferred region.

The above-mentioned preferred regions set out in FIG. 17 include, butare not limited to, regions of the aforementioned types identified byanalysis of the amino acid sequence set out in FIGS. 1A-C. As set out inFIG. 17, such preferred regions include Garnier-Robson alpha-regions,beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions,beta-regions, and coil-regions, Kyte-Doolittle hydrophilic regions andhydrophobic regions, Eisenberg alpha- and beta-amphipathic regions,Karplus-Schulz flexible regions, Emini surface-forming regions andJameson-Wolf regions of high antigenic index.

Among highly preferred fragments in this regard are those that comprisereigons of TNF-gamma-alpha and/or TNF-gamma-beta that combine severalstructural features, such as several (e.g., 1, 2, 3 or 4) of thefeatures set out above.

Additional preferred nucleic acid fragments of the present inventioninclude nucleic acid molecules encoding one or more epitope-bearingportions of the TNF-gamma polypeptide. In particular, such nucleic acidfragments of the present invention included nucleic acid moleculesencoding: a polypeptide comprising amino acid residues from about Thr-24to about Asn-32 in SEQ ID NO:2; a polypeptide comprising amino acidresidues from about Ile-37 to about Ile-45 in SEQ ID NO:2; a polypeptidecomprising amino acid residues from about Met-54 to about Arg-62 in SEQID NO:2; a polypeptide comprising amino acid residues from about Gln-63to about Asp-71 in SEQ ID NO:2; a polypeptide comprising amino acidresidues from about Glu-57 to about Gly-65 in SEQ ID NO:2; a polypeptidecomprising amino acid residues from about Val-80 to about Thr-88 in SEQID NO:2; a polypeptide comprising amino acid residues from about Leu-116to about Val-124 in SEQ ID NO:2; and a polypeptide comprising amino acidresidues from about Asp-133 to about Phe-141 in SEQ ID NO:2. Thesepolypeptide fragments have been determined to bear antigenic epitopes ofthe TNF-gamma protein by the analysis of the Jameson-Wolf antigenicindex, as shown in FIG. 17, above. Methods for determining other suchepitope-bearing portions of TNF-gamma are described in detail below.

Polypeptide fragments which bear antigenic epitopes of theTNF-gamma-beta protein may be easily determined by one of skill in theart using the above-described analysis of the Jameson-Wolf antigenicindex, as shown in FIG. 17. Methods for determining other suchepitope-bearing portions of TNF-gamma-beta are described in detailbelow.

Another embodiment of the invention is directed to polynucleotides thathybridize, preferably under stringent hybridization conditions, to aportion of the polynucleotide sequence of a polynucleotide of theinvention such as, for instance, the cDNA clone contained in ATCCDeposit No. 75927, the cDNA clone contained in ATCC Deposit 203055 or aTNF-gamma polynucleotide fragment as described herein. By “stringenthybridization conditions” is intended overnight incubation at 42° C. ina solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1×SSC at about 65° C. Bya polynucleotide which hybridizes to a “portion” of a polynucleotide isintended a polynucleotide (either DNA or RNA) hybridizing to at least 15nucleotides (nt), and more preferably at least 20 nt, still morepreferably at least 30 nt, and even more preferably 30-70, or 80-150 nt,or the entire length of the reference polynucleotide. These are usefulas diagnostic probes and primers as discussed above and in more detailbelow. Of course, a polynucleotide which hybridizes only to a poly Asequence (such as the 3′ terminal poly tract of the TNF-gamma cDNA shownin SEQ ID NO:1 or SEQ ID NO:19), or to a complementary stretch of T (orU) residues, would not be included in a polynucleotide of the inventionused to hybridize to a portion of a nucleic acid of the invention, sincesuch a polynucleotide would hybridize to any nucleic acid moleculecontaining a poly (A) stretch or the complement thereof (e.g.,practically any double-stranded cDNA clone generated using oligo dT as aprimer).

In preferred embodiments, polynucleotides which hybridize to thereference polynucleotides disclosed herein encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the polynucleotide sequences depictedin FIGS. 1A-C (SEQ ID NO:1) and/or FIGS. 20A and B (SEQ ID NO:19), orthe cDNAs contained in the deposit.

Alternative embodiments are directed to polynucleotides which hybridizeto the reference polynucleotide (i.e., a polynucleotide sequencedisclosed herein), but do not retain biological activity. While thesepolynucleotides do not retain biological activity, they have uses, suchas, for example, as probes for the polynucleotide of SEQ ID NO:1, forrecovery of the polynucleotide, as diagnostic probes, and as PCRprimers.

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs orderivatives of the TNF-gamma protein. Variants may occur naturally, suchas a natural allelic variant. By an “allelic variant” is intended one ofseveral alternate forms of a gene occupying a given locus on achromosome of an organism (Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985)). Non-naturally occurring variants may be produced usingart-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions,deletions or additions of the polynucleotide sequences described herein(including fragments). The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingregions, non-coding regions, or both. Alterations in the coding regionsmay produce conservative or nonconservative amino acid substitutions,deletions or additions. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the TNF-gamma protein or portions thereof.Also especially preferred in this regard are conservative substitutions.

Further embodiments of the invention are directed to isolated nucleicacid molecules comprising a polynucleotide sequence at least 70% or atleast 80% identical, more preferably at least 90% identical, and stillmore preferably at least 95%, 96%, 97%, 98% or 99% identical to apolynucleotide having a nucleotide sequence selected from the groupconsisting of: (a) a nucleotide sequence encoding the TNF-gamma-alphapolypeptide having the complete amino acid sequence in SEQ ID NO:2(i.e., positions −27 to 147 of SEQ ID NO:2); (b) a nucleotide sequenceencoding the TNF-gamma-alpha polypeptide having the complete amino acidsequence in SEQ ID NO:2 excepting the N-terminal methionine (i.e.,positions −26 to 147 of SEQ ID NO:2); (c) a nucleotide sequence encodingthe mature TNF-gamma-alpha polypeptide having the amino acid sequence inSEQ ID NO:2 shown as positions 1 to 147 of SEQ ID NO:2; (d) a nucleotidesequence encoding the extracellular domain of the TNF-gamma-alphapolypeptide having the amino acid sequence in SEQ ID NO:2 shown aspositions 1 to 147 of SEQ ID NO:2; (e) a nucleotide sequence encodingthe TNF-gamma-alpha polypeptide having the complete amino acid sequenceencoded by the cDNA clone: HUVEO91 contained in ATCC Deposit No. 75927;(f) a nucleotide sequence encoding the TNF-gamma-alpha polypeptidehaving the complete amino acid sequence excepting the N-terminalmethionine encoded by the cDNA clone HUVEO91 contained in ATCC DepositNo. 75927; (g) a nucleotide sequence encoding the mature TNF-gamma-alphapolypeptide having the amino acid sequence encoded by the cDNA cloneHUVEO91 contained in ATCC Deposit No. 75927; (h) a nucleotide sequenceencoding the extracellular domain of the TNF-gamma-alpha polypeptidehaving the amino acid sequence encoded by the cDNA clone HUVEO91contained in ATCC Deposit No. 75927; (i) a nucleotide sequence encodinga polypeptide fragment described herein; and (j) a nucleotide sequencecomplementary to any of the nucleotide sequences in (a), (b), (c), (d),(e), (f), (g), (h) or (i), above. The polypeptides of the presentinvention also include polypeptides having an amino acid sequence atleast 80% identical, more preferably at least 90% identical, and stillmore preferably 95%, 96%, 97%, 98% or 99% identical to those describedin (a), (b), (c), (d), (e), (f), (g), (h), (i) or (j), above, as well aspolypeptides having an amino acid sequence with at least 90% similarity,and more preferably at least 95% similarity, to those above.

Further embodiments of the invention are directed to isolated nucleicacid molecules comprising a polynucleotide sequence at least 70% or atleast 80% identical, more preferably at least 90% identical, and stillmore preferably at least 95%, 96%, 97%, 98% or 99% identical to apolynucleotide having a nucleotide sequence selected from the groupconsisting of: (a) a nucleotide sequence encoding the TNF-gamma-betapolypeptide having the complete amino acid sequence in SEQ ID NO:20(i.e., positions 1 to 251 of SEQ ID NO:20); (b) a nucleotide sequenceencoding the TNF-gamma-beta polypeptide having the complete amino acidsequence in SEQ ID NO:20 excepting the N-terminal methionine (i.e.,positions 2 to 251 of SEQ ID NO:20); (c) a nucleotide sequence encodingthe mature TNF-gamma-beta polypeptide having the amino acid sequence inSEQ ID NO:20 shown as positions 62 to 251 of SEQ ID NO:20; (d) anucleotide sequence encoding the intracellular domain of theTNF-gamma-beta polypeptide having the amino acid sequence in SEQ IDNO:20 shown as positions 1 to 35 of SEQ ID NO:20; (e) a nucleotidesequence encoding the extracellular domain of the TNF-gamma-betapolypeptide having the amino acid sequence in SEQ ID NO:20 shown aspositions 62 to 251 of SEQ ID NO:20; (f) a nucleotide sequence encodingthe TNF-gamma-beta polypeptide having the complete amino acid sequenceencoded by the cDNA clone HEMCZ56 contained in ATCC Deposit No. 203055;(g) a nucleotide sequence encoding the TNF-gamma-beta polypeptide havingthe complete amino acid sequence excepting the N-terminal methionineencoded by the cDNA clone HEMCZ56 contained in ATCC Deposit No. 203055;(h) a nucleotide sequence encoding the mature TNF-gamma-beta polypeptidehaving the amino acid sequence encoded by the cDNA clone HEMCZ56contained in ATCC Deposit No. 203055; (i) a nucleotide sequence encodingthe intracellular domain of the TNF-gamma-beta polypeptide having theamino acid sequence encoded by the cDNA clone HEMCZ56 contained in ATCCDeposit No. 203055; (j) a nucleotide sequence encoding the extracellulardomain of the TNF-gamma-beta polypeptide having the amino acid sequenceencoded by the cDNA clone HEMCZ56 contained in ATCC Deposit No. 203055;and (k) a nucleotide sequence complementary to any of the nucleotidesequences in (a), (b), (c), (d), (e), (f), (g), (h), (i) or (j), above.The polypeptides of the present invention also include polypeptideshaving an amino acid sequence at least 80% identical, more preferably atleast 90% identical, and still more preferably 95%, 96%, 97%, 98% or 99%identical to those described in (a), (b), (c), (d), (e), (f), (g), (h),(i), (j) or (k), above, as well as polypeptides having an amino acidsequence with at least 90% similarity, and more preferably at least 95%similarity, to those above.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence of the presentinvention, it is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding theTNF-gamma polypeptide. In other words, to obtain a polynucleotide havinga nucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. The reference (query)sequence may be the entire nucleotide sequence shown in FIGS. 1A-C (SEQID NO:1) and FIGS. 20A and B (SEQ ID NO:19), or any fragment asdescribed herein.

As a practical matter, whether any particular nucleic acid molecule isat least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to, forinstance, the nucleotide sequence shown in FIGS. 1A-C (SEQ ID NO:1),FIGS. 20A and B (SEQ ID NO:19), or to the nucleotide sequence of thedeposited cDNA clones can be determined conventionally using knowncomputer programs such as the Bestfit program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).Bestfit uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2:482-489 (1981), to find the bestsegment of homology between two sequences. When using Bestfit or anyother sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference nucleotide sequence and that gaps in homology of up to5% of the total number of nucleotides in the reference sequence areallowed.

In a specific embodiment, the identity between a reference (query)sequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, is determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDBalignment of DNA sequences to calculate percent identity are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty 0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter. According to this embodiment, if thesubject sequence is shorter than the query sequence because of 5′ or 3′deletions, not because of internal deletions, a manual correction ismade to the results to take into consideration the fact that the FASTDBprogram does not account for 5′ and 3′ truncations of the subjectsequence when calculating percent identity. For subject sequencestruncated at the 5′ or 3′ ends, relative to the query sequence, thepercent identity is corrected by calculating the number of bases of thequery sequence that are 5′ and 3′ of the subject sequence, which are notmatched/aligned, as a percent of the total bases of the query sequence.A determination of whether a nucleotide is matched/aligned is determinedby results of the FASTDB sequence alignment. This percentage is thensubtracted from the percent identity, calculated by the above FASTDBprogram using the specified parameters, to arrive at a final percentidentity score. This corrected score is what is used for the purposes ofthis embodiment. Only bases outside the 5′ and 3′ bases of the subjectsequence, as displayed by the FASTDB alignment, which are notmatched/aligned with the query sequence, are calculated for the purposesof manually adjusting the percent identity score. For example, a 90 basesubject sequence is aligned to a 100 base query sequence to determinepercent identity. The deletions occur at the 5′ end of the subjectsequence and therefore, the FASTDB alignment does not show amatched/alignment of the first 10 bases at 5′ end. The 10 unpaired basesrepresent 10% of the sequence (number of bases at the 5′ and 3′ ends notmatched/total number of bases in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 bases were perfectly matched the finalpercent identity would be 90%. In another example, a 90 base subjectsequence is compared with a 100 base query sequence. This time thedeletions are internal deletions so that there are no bases on the 5′ or3′ of the subject sequence which are not matched/aligned with the query.In this case the percent identity calculated by FASTDB is not manuallycorrected. Once again, only bases 5′ and 3′ of the subject sequencewhich are not matched/aligned with the query sequence are manuallycorrected for. No other manual corrections are made for the purposes ofthis embodiment.

In further embodiments, the present invention is directed topolynucleotides having at least a 70% identity, preferably at least 90%and more preferably at least a 95% identity to a polynucleotide whichencodes the polypeptide of SEQ ID NO:2 as well as fragments thereof,which fragments have at least 30 bases and preferably at least 50 basesand to polypeptides encoded by such polynucleotides.

In further embodiments, the present invention is directed topolynucleotides having at least a 70% identity, preferably at least 90%and more preferably at least a 95% identity to a polynucleotide whichencodes the polypeptide of SEQ ID NO:20 as well as fragments thereof,which fragments have at least 30 bases and preferably at least 50 basesand to polypeptides encoded by such polynucleotides.

The present application is directed to nucleic acid molecules at least70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotidesequence shown in FIGS. 1A-C (SEQ ID NO:1), FIGS. 20A and B (SEQ IDNO:19), or to the nucleic acid sequence of the deposited cDNA clones, orfragments thereof, irrespective of whether they encode a polypeptidehaving TNF-gamma functional activity. This is because even where aparticular nucleic acid molecule does not encode a polypeptide havingTNF-gamma functional activity, one of skill in the art would still knowhow to use the nucleic acid molecule, for instance, as a hybridizationprobe or a polymerase chain reaction (PCR) primer. Uses of the nucleicacid molecules of the present invention that do not encode a polypeptidehaving TNF-gamma functional activity include, inter alia, (1) isolatingthe TNF-gamma gene or allelic variants thereof in a cDNA library; (2) insitu hybridization (e.g., “FISH”) to metaphase chromosomal spreads toprovide precise chromosomal location of the TNF-gamma gene, as describedin Verma et al., Human Chromosomes: A Manual of Basic Techniques,Pergamon Press, N.Y. (1988); and (3) Northern Blot analysis fordetecting TNF-gamma mRNA expression in specific tissues.

Preferred, however, are nucleic acid molecules having sequences at least70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequence shown in FIGS. 1A-C (SEQ ID NO:1), FIGS. 20A and B (SEQ IDNO:19), or to the nucleic acid sequence of the deposited cDNA clones, orfragments thereof, which do, in fact, encode a polypeptide havingTNF-gamma functional activity. By “a polypeptide having TNF-gammafunctional activity” is intended polypeptides exhibiting activitysimilar, but not necessarily identical, to an activity of the TNF-gammapolypeptide of the invention (either the fall-length protein or,preferably, the mature protein), as measured in a particular immunoassayand/or biological assay. For example, TNF-gamma activity can be measuredusing an apoptosis assay as described in Example 7, by determining therelative ability of TNF-gamma to inhibit the FGF-2-induced formation ofcapillary-like tubular structure formation in cultures of ABAE cells asdescribed in detail in Example 9 or in a chorioallantoic membrane (CAM)angiogenesis assay as described in Example 10, by its effect(s) on theactivation of cellular NF-kB and c-Jun kinase (JNK) as described inExample 12, and in several additional ways described in the remainingExamples and in the art.

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 70%, 80%, 90%, 95%,96%, 97%, 98%, or 99% identical to the nucleic acid sequence of thedeposited cDNA or the nucleic acid sequence shown in FIGS. 1A-C (SEQ IDNO:1), FIGS. 20A and 20B (SEQ ID NO:19), or fragments thereof, willencode a polypeptide “having TNF-gamma activity.” In fact, sincedegenerate variants of these nucleotide sequences all encode the samepolypeptide, in many instances, this will be clear to the skilledartisan even without performing the above described assay. It will befurther recognized in the art that, for such nucleic acid molecules thatare not degenerate variants, a reasonable number will also encode apolypeptide having TNF-gamma activity. This is because the skilledartisan is fully aware of amino acid substitutions that are either lesslikely or not likely to significantly effect protein function (e.g.,replacing one aliphatic amino acid with a second aliphatic amino acid).For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in J. U. Bowie et al., “Deciphering theMessage in Protein Sequences: Tolerance to Amino Acid Substitutions,”Science 247:1306-1310 (1990), wherein the authors indicate that proteinsare surprisingly tolerant of amino acid substitutions.

Additional embodiments of the invention are directed to isolated nucleicacid molecules comprising a polynucleotide which encodes the amino acidsequence of a TNF-gamma polypeptide (e.g., a TNF-gamma polypeptidefragment described herein) having an amino acid sequence which containsat least one conservative amino acid substitution, but not more than 50conservative amino acid substitutions, even more preferably, not, morethan 40 conservative amino acid substitutions, still more preferably,not more than 30 conservative amino acid substitutions, and still evenmore preferably, not more than 20 conservative amino acid substitutions,10-20 conservative amino acid substitutions, 5-10 conservative aminoacid substitutions, 1-5 conservative amino acid substitutions, 3-5conservative amino acid substitutions, or 1-3 conservative amino acidsubstitutions. Of course, in order of ever-increasing preference, it ishighly preferable for a polynucleotide which encodes the amino acidsequence of a TNF-gamma polypeptide to have an amino acid sequence whichcontains not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservativeamino acid substitutions.

Polynucleotide Assays

The invention also encompasses the use of TNF-gamma polynucleotides todetect complementary polynucleotides, such as, for example, as adiagnostic reagent for detecting diseases or susceptibility to diseasesrelated to the presence of mutated TNF-gamma-alpha or TNF-gamma-beta.Such diseases are related to an under-expression of TNF-gamma-alpha orTNF-gamma-beta, such as, for example, abnormal cellular proliferationsuch as tumors and cancers.

Individuals carrying mutations in the human TNF-gamma gene may bedetected at the DNA level by a variety of techniques. Nucleic acids fordiagnosis may be obtained from a patient's cells, such as from blood,urine, saliva, tissue biopsy and autopsy material. The genomic DNA maybe used directly for detection or may be amplified enzymatically byusing PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analysis.RNA or cDNA may also be used for the same purpose. As an example, PCRprimers complementary to the nucleic acid encoding TNF-gamma-alpha orTNF-gamma-beta can be used to identify and analyze TNF-gamma mutations.For example, deletions and insertions can be detected by a change insize of the amplified product in comparison to the normal genotype.Point mutations can be identified by hybridizing amplified DNA toradiolabeled TNF-gamma RNA or alternatively, radiolabeled TNF-gammaantisense DNA sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al, Science, 230:1242 (1985)).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

The deposit(s) referred to herein will be maintained under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicro-organisms for purposes of Patent Procedure. These deposits areprovided merely as convenience to those of skill in the art and are notan admission that a deposit is required under 35 U.S.C. § 112. Thesequence of the polynucleotides contained in the deposited materials, aswell as the amino acid sequence of the polypeptides encoded thereby, areincorporated herein by reference and are controlling in the event of anyconflict with any description of sequences herein. A license may berequired to make, use or sell the deposited materials, and no suchlicense is hereby granted.

Vectors and Host Cells

The present invention also relates to vectors which include the isolatedpolynucleotides of the present invention, host cells which aregenetically engineered with the recombinant vectors, or which areotherwise engineered to produce the polypeptides of the invention, andthe production of polypeptides of the invention by recombinanttechniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the TNF-gamma genes. The culture conditions,such as temperature, pH and the like, are those previously used with thehost cell selected for expression, and will be apparent to theordinarily skilled artisan.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operably associated with anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli lac or trp, the phagelambda P promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila S2 and Sf9;animal cells such as CHO, COS or Bowes melanoma, adenoviruses, plantcells, etc. The selection of an appropriate host is deemed to be withinthe scope of those skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably associated with thesequence. Large numbers of suitable vectors and promoters are known tothose of skill in the art, and are commercially available. The followingvectors are provided by way of example. Bacterial: pHE4-5 (ATCCAccession No. 209311; and variations thereof), pQE70, pQE60, pQE-9(Qiagen), pBS, pD10, phagescript, psiX174, pBluescript SK, pbsks, pNH8A,pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any otherplasmid or vector may be used as long as they are replicable and viablein the host.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are PKK232-8 and PCM7. Particular namedbacterial promoters include lac1, lacZ, T3, T7, gpt, lambda P, P, andtrp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retroviruses, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation. (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, (1986)).

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered, to delete or replace endogenousgenetic material (e.g., TNF-gamma coding sequence), and/or to includegenetic material (e.g., heterologous polynucleotide sequences) that isoperably associated with TNF-gamma polynucleotides of the invention, andwhich activates, alters, and/or amplifies endogenous TNF-gammapolynucleotides. For example, techniques known in the art may be used tooperably associate heterologous control regions (e.g., promoter and/orenhancer) and endogenous TNF-gamma polynucleotide sequences viahomologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issuedJun. 24, 1997; International Publication No. WO 96/29411, published Sep.26, 1996; International Publication No. WO 94/12650, published Aug. 4,1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); andZijistra et al., Nature 342:435-438 (1989), the disclosures of each ofwhich are incorporated by reference in their entireties).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), thedisclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), alpha-factor acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, for example, stabilization or simplifiedpurification of expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium, and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative, but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period. Cells aretypically harvested by centrifugation, disrupted by physical or chemicalmeans, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman (Cell23:175 (1981)), and other cell lines capable of expressing a compatiblevector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.Mammalian expression vectors will comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation site, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking nontranscribedsequences. DNA sequences derived from the SV40 splice, andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements.

The TNF-gamma polypeptides can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the mature protein. Finally,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

The invention encompasses TNF-gamma-alpha and TNF-gamma-betapolypeptides which are differentially modified during or aftertranslation, e.g., by glycosylation, acetylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to an antibody molecule or other cellularligand, etc. Any of numerous chemical modifications may be carried outby known techniques, including but not limited, to specific chemicalcleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH₄; acetylation, formylation, oxidation, reduction;metabolic synthesis in the presence of tunicamycin; etc.

In addition, polypeptides of the invention can be chemically synthesizedusing techniques known in the art. For example, a peptide correspondingto a fragment of the TNF-gamma-alpha and TNF-gamma-beta polypeptides ofthe invention can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into the latssequence. Non-classical amino acids include but are not limited to, tothe D-isomers of the common amino acids, 2,4-diaminobutyric acid,a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid,g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid,3-amino propionic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,b-alanine, fluoro-amino acids, designer amino acids such as b-methylamino acids, Ca-methyl amino acids, Na-methyl amino acids, and aminoacid analogs in general. Furthermore, the amino acid can be D(dextrorotary) or L (levorotary).

At least fifteen TNF-gamma-alpha expression constructs have beengenerated by the inventors herein to facilitate the production ofTNF-gamma polypeptides of several sizes and in several systems. Ofthese, four have been constructed which encode a full-length TNF-gammapolypeptide. The full-length constructs are: (i) pQE9TNFg-27/147, (ii)pQE70TNFg, (iii) pC1TNFg, and pcDNA3TNFg. In the case of the firstexpression construct listed (pQE9TNFg-27/147), the construct was used toproduce a full-length TNF-gamma-alpha polypeptide with an N-terminal sixhistidine amino acid tag according to the method of Example 1. Afull-length TNF-gamma-alpha polypeptide lacking the histidine tag wasproduced in bacteria by using the pQE70TNFg construct essentially as wasdone in Example 1. In addition, a full-length TNF-gamma-alphapolypeptide lacking a histidine tag was produced in mammalian cells byusing either the pC1TNFg or pcDNA3TNFg constructs according to themethod of Example 3. Further, the mature TNF-gamma-alpha polypeptide wasproduced and secreted from mammalian cells under direction of theinterleukin (IL)-6 signal peptide from a construct designatedpcDNA3/IL6TNFg-1/149 (see Example 11).

The remaining TNF-gamma-alpha expression constructs were used to expressvarious TNF-gamma muteins from bacterial, baculoviral, and mammaliansystems. Four N-terminal deletion mutations have been generated usingthe pQE60 bacterial expression vector. These N-terminal deletionmutation constructs are: (i) pQE60TNFg-31147 (representing a possiblemature TNF-gamma polypeptide;

the polypeptide expressed by this construct is identical to amino acidresidues 107-251 of the TNF-gamma-beta of SEQ ID NO:20), (ii)pQE60TNFg12/147 (representing amino acid residues 12-147 of SEQ ID NO:2and residues 116-251 of SEQ ID NO:20), (iii) pQE60TNFg22/147(representing amino acid residues 22-147 of SEQ ID NO:2 and residues126-251 of SEQ ID NO:20), and (iv) pQE60TNFg28/147 (representing aminoacid residues 28-147 of SEQ ID NO:2 and residues 132-251 of SEQ IDNO:20). Each of these expression constructs can be used to produce aTNF-gamma polypeptide in a bacterial system which exhibits an N-terminaldeletion of 29, 38, 48 and 54 amino acids, respectively, with regard tothe full-length TNF-gamma-alpha polypeptide or an N-terminal deletion of106, 115, 125, and 131 amino acids, respectively, with regard to thefull-length TNF-gamma-beta polypeptide.

Further N-terminal deletion mutation bacterial expression constructshave been generated. A construct designated pHE4 VEGI T30-L174 has beengenerated using the bacterial expression vector pHE4 to express aminoacids threonine-30 to leucine-174 of the TNF-gamma-alpha sequence shownin FIGS. 1A-C (residues threonine-3 to leucine-147 of SEQ ID NO:2) whichcorrespond exactly to amino acid residues threonine-107 to leucine-251of the TNF-gamma-beta sequence shown in FIGS. 20A and 20B (residuesthreonine-107 to leucine-251 of SEQ ID NO:20). Additional bacterialexpression constructs generated include pQE9.VEGI.his.T28-L174,pHE4.VEGI.T28-L174, pHE4.VEGI.T51-L174, and pHE4.VEGI.T58-L174. Theseconstructs are based on either the pQE9 or pHE4 bacterial expressionvectors. The construct designations indicate the expression vector, thegene name, and the amino acid residues expressed by the construct (e.g.pQE9.VEGI.T28-L174 indicates that the pQE9 bacterial expression vectoris used to express amino acids threonine (T)-28 through leucine (L)-174of the TNF-gamma-alpha polypeptide (VEGI is a laboratory designation forTNF-gamma-alpha)).

A TNF-gamma expression construct has been generated which can be used toproduce a secreted mature TNF-gamma polypeptide from a mammalian system.The construct has been designated pC1/IL6TNFg-3/147. It encodes thesignal peptide from the human IL-6 gene fused to the mature TNF-gammasequence. A similar construct has been generated which contains theCK-beta8 signal peptide (amino acids −21 to −1 of the CK-beta8 sequencedisclosed in published PCT application PCT/US95/09058; filed Jun. 23,1995) fused to the amino terminus of amino acids 12-149 ofTNF-gamma-alpha (SEQ ID NO:2; that is, amino acids 116-251 ofTNF-gamma-beta (SEQ ID NO:20)) in the context of the pC4 mammalianexpression vector. This construct has been designatedpC4/CK-beta8TNFg-12/147. A variant of this construct has been generatedwhich can be used to express amino acids 12-147 of TNF-gamma fused tothe human IgG Fc region at the TNF-gamma carboxy terminus. This fusionprotein is also secreted under the direction of the CK-beta8 signalpeptide and has been designated pC4/CK-beta8TNFg-12/147Fc. The sequenceof the human Fc portion of the fusion molecule is shown in SEQ ID NO:18.Other sequences could be used which are known to those of skill in theart.

Amino acids −3 to 147 of TNF-gamma-alpha (SEQ ID NO:2; which correspondto amino acid residues 102 to 251 of TNF-gamma-beta (SEQ ID NO:20)) canbe expressed and secreted from a baculovirus system by using a constructdesignated pA2GPTNFg-3/147. This expression construct encodes the matureTNF-gamma coding sequence fused at its amino terminus to the baculoviralGP signal peptide.

Two retroviral TNF-gamma expression constructs have also been generated.The first of these has been designated pG1SamEN/TNFg-3/149. Thisexpression construct can be used to produce full-length TNF-gammaprotein from a mammalian system. A related construct,pG1SamEN/CK-beta8TNFg-12/149, has been generated which can be used toproduce and secrete mature TNF-gamma protein from a mammalian systemunder the direction of the CK-beta8 signal peptide.

Further polypeptides of the present invention include polypeptides whichhave at least 90% similarity, more preferably at least 95% similarity,and still more preferably at least 96%, 97%, 98% or 99% similarity tothose described above. The polypeptides of the invention also comprisethose which are at least 80% identical, more preferably at least 90% or95% identical, still more preferably at least 96%, 97%, 98% or 99%identical to the polypeptide encoded by the deposited cDNA or to thepolypeptide of SEQ ID NO:2, and also include portions of suchpolypeptides with at least 30 amino acids and more preferably at least50 amino acids.

Polypeptides and Fragments

The present invention further relates to an isolated TNF-gamma-alphapolypeptide which has the deduced amino acid sequence of FIGS. 1A-C (SEQID NO:2) or which has the amino acid sequence encoded by the depositedcDNA HUVEO91, as well as fragments, analogs and derivatives of suchpolypeptide.

The present invention also relates to a TNF-gamma-beta polypeptide whichhas the deduced amino acid sequence of FIGS. 20A and 20B (SEQ ID NO:20)or which has the amino acid sequence encoded by the deposited cDNAHEMCZ56, as well as fragments, analogs and derivatives of suchpolypeptide.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified toa point within the range of near complete (e.g., >90% pure) to complete(e.g., >99% pure) homogeneity. The term “isolated” means that thematerial is removed from its original environment (e.g., the naturalenvironment if it is naturally occurring). For example, anaturally-occurring polynucleotide or polypeptide present in a livinganimal is not isolated, but the same polynucleotide or polypeptide,separated from some or all of the coexisting materials in the naturalsystem, is isolated. Also intended as an “isolated polypeptide” arepolypeptides that have been purified partially or substantially from arecombinant host cell. For example, a recombinantly produced version ofa TNF-gamma polypeptide can be substantially purified by the one-stepmethod described by Smith and Johnson (Gene 67:31-40 (1988)). Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.Isolated polypeptides and polynucleotides according to the presentinvention also include such molecules produced naturally orsynthetically. Polypeptides and polynucleotides of the invention alsocan be purified from natural or recombinant sources using anti-TNF-gammaantibodies of the invention in methods which are well known in the artof protein purification.

The terms “fragment,” “derivative” and “analog” when referring to thepolypeptides of FIGS. 1A-C or FIGS. 20A and 20B, and those polypeptodesencoded by the deposited cDNAs, means a polypeptide which retains aTNF-gamma functional activity, i.e., displays one or more functionalactivities associated with a full-length and/or mature TNF-gammapolypeptide disclosed in FIGS. 1A-C (SEQ ID NO:2), FIGS. 20A and B (SEQID NO:20), and/or encoded by one or both of the deposited clones(HUVEO91 and HEMCZ56). As one example, such fragments, derivatives, oranalogs, which have the desired immunogenicity or antigenicity can beused, for example, in immunoassays, for immunization, for inhibition ofTNF-gamma activity, etc. Thus, a specific embodiment of the inventionrelates to a TNF-gamma fragment that can be bound by an antibody thatspecifically binds the TNF-gamma by polypeptide sequence disclosed inFIGS. 1A-C (SEQ ID NO:2), FIGS. 20 A and B (SEQ ID NO:20)), and/or whichis encoded by one or both of the deposited clones (HUVEO91 and HEMCZ56).

As another example, TNF-gamma fragments, derivatives or analogs whichhave TNF-gamma biological activity (e.g., a mature TNF-gamma-alphapolypeptide or the extracellular domain of a TNF-gamma-beta polypeptide)are provided. TNF-gamma fragments, derivatives, and analogs that retain,or alternatively lack a desired TNF-gamma property of interest (e.g.,inhibition of cell proliferation, tumor inhibition, inhibition ofangiogenesis, anti-arthritis by the inhibition of angiogenesis and/orendothelial cell proliferation associated with invading pannus in boneand cartilage, an inducer of NF-κB and c-Jun kinase (JNK), an inducer ofcell adhesion, and as an inducer apoptosis (See Examples, particularlyExamples 12-15)) can be used as inducers or inhibitors, respectively, ofsuch properties and its physiological correlates.

The polypeptides of the invention may exist as a membrane bound receptorhaving a transmembrane region and an intra- and extracellular region orthey may exist in soluble form wherein the transmembrane domain islacking. One example of such a form of TNF-gamma is the TNF-gamma-betapolypeptide sequence shown in FIGS. 20A and B (SEQ ID NO:20) whichcontains a transmembrane, intracellular and extracellular domain.

It will be recognized in the art that some amino acid sequences of theTNF-gamma polypeptide can be varied without significant effect of thestructure or function of the protein. If such differences in sequenceare contemplated, it should be remembered that there will be criticalareas on the protein which determine activity. Thus, the inventionfurther includes variations of the TNF-gamma polypeptide which showsubstantial TNF-gamma polypeptide activity or which include regions ofTNF-gamma protein such as the polypeptide fragments disclosed herein.Such variants include deletions, insertions, inversions, repeats, andtype substitutions selected according to general rules known in the artso as have little effect on activity. For example, guidance concerninghow to make phenotypically silent amino acid substitutions is providedwherein the authors indicate that there are two main approaches forstudying the tolerance of an amino acid sequence to change (Bowie etal., Science 247:1306-1310 (1990)). The first method relies on theprocess of evolution, in which mutations are either accepted or rejectedby natural selection. The second approach uses genetic engineering tointroduce amino acid changes at specific positions of a cloned gene andselections or screens to identify sequences that maintain functionality.As the authors state, these studies have revealed that proteins aresurprisingly tolerant of amino acid substitutions. The authors furtherindicate which amino acid changes are likely to be permissive at acertain position of the protein. For example, most buried amino acidresidues require nonpolar side chains, whereas few features of surfaceside chains are generally conserved. Other such phenotypically silentsubstitutions are described by Bowie and coworkers (supra) and thereferences cited therein. Typically seen as conservative substitutionsare the replacements, one for another, among the aliphatic amino acidsAla, Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr.

Thus, the fragment, derivative or analog of the polypeptide of SEQ IDNO:2, or of SEQ ID NO:20, or those encoded by the deposited cDNAs, maybe (i) one in which one or more of the amino acid residues aresubstituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature form of theTNF-gamma polypeptide is fused with another compound, such as a compoundto increase the half-life of the polypeptide (for example, polyethyleneglycol), or (iv) one in which the additional amino acids are fused tothe above form of the polypeptide, such as an IgG Fc fusion regionpeptide or leader or secretory sequence or a sequence which is employedfor purification of the above form of the polypeptide or a proproteinsequence. Such fragments, derivatives and analogs are deemed to bewithin the scope of those skilled in the art from the teachings herein.

Thus, the TNF-gamma of the present invention may include one or moreamino acid substitutions, deletions or additions, either from naturalmutations or human manipulation. As indicated, changes are preferably ofa minor nature, such as conservative amino acid substitutions that donot significantly affect the folding or activity of the protein (seeTable 1).

TABLE 1 Conservative Amino Acid Substitutions. Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

Embodiments of the invention are directed to polypeptides which compriseamino acid sequence of a TNF-gamma polypeptide described herein, buthaving an amino acid sequence which contains at least one conservativeamino acid substitution, but not more than 50 conservative amino acidsubstitutions, even more preferably, not more than 40 conservative aminoacid substitutions, still more preferably, not more than 30 conservativeamino acid substitutions, and still even more preferably, not more than20 conservative amino acid substitutions, when compared with theTNF-gamma polynucleotide sequence described herein. Of course, in orderof ever-increasing preference, it is highly preferable for a peptide orpolypeptide to have an amino acid sequence which comprises the aminoacid sequence of a TNF-gamma polypeptide, which contains at least one,but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative aminoacid substitutions.

In further specific embodiments, the number of substitutions, additionsor deletions in the amino acid sequence of FIGS. 1A-C (SEQ ID NO:2),FIGS. 20 A and B (SEQ ID NO:20), a polypeptide sequence encoded by thedeposited clones, and/or any of the polypeptide fragments describedherein (e.g., the extracellular domain or intracellular domain) is 75,70, 60, 50, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or150-50, 100-50, 50-20, 30-20, 20-15, 20-10, 15-10, 10-1, 5-10, 1-5, 1-3or 1-2.

To improve or alter the characteristics of TNF-gamma polypeptides,protein engineering may be employed. Recombinant DNA technology known tothose skilled in the art can be used to create novel mutant proteins ormuteins including single or multiple amino acid substitutions,deletions, additions or fusion proteins. Such modified polypeptides canshow, e.g., enhanced activity or increased stability. In addition, theymay be purified in higher yields and show better solubility than thecorresponding natural polypeptide, at least under certain purificationand storage conditions.

Thus, the invention also encompasses TNF-gamma derivatives and analogsthat have one or more amino acid residues deleted, added, or substitutedto generate TNF-gamma polypeptides that are better suited forexpression, scale up, etc., in the host cells chosen. For example,cysteine residues can be deleted or substituted with another amino acidresidue in order to eliminate disulfide bridges; N-linked glycosylationsites can be altered or eliminated to achieve, for example, expressionof a homogeneous product that is more easily recovered and purified fromyeast hosts which are known to hyperglycosylate N-linked sites. To thisend, a variety of amino acid substitutions at one or both of the firstor third amino acid positions on any one or more of the glycosylationrecognitions sequences in the TNF-gamma polypeptides of the invention,and/or an amino acid deletion at the second position of any one or moresuch recognition sequences will prevent glycosylation of the TNF-gammapolypeptide at the modified tripeptide sequence (see, e.g., Miyajimo etal., EMBO J 5(6):1193-1197).

Amino acids in the TNF-gamma protein of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as receptor binding or in vitro proliferative activity.

Of special interest are substitutions of charged amino acids with othercharged or neutral amino acids which may produce proteins with highlydesirable improved characteristics, such as less aggregation.Aggregation may not only reduce activity but also be problematic whenpreparing pharmaceutical formulations, because aggregates can beimmunogenic (Pinckard, et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins, et al., Diabetes 36:838-845 (1987); Cleland, et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993)).

Since TNF-gamma is a member of the TNF-related protein family, tomodulate rather than completely eliminate biological activities ofTNF-gamma preferably additions, substitutions, or deletions are made insequences encoding amino acids in the conserved TNF-like domain, i.e.,in positions 17-147 of SEQ ID NO:2 or positions 121-251 of SEQ ID NO:20,more preferably in residues within this region which are not conservedin all members of the TNF-related protein family (see FIGS. 2A-2C). Alsoforming part of the present invention are isolated polynucleotidescomprising nucleic acid sequences which encode the above TNF-gammavariants.

Several amino acids of the TNF-gamma polypeptide are highly conservedacross the known members of the TNF-related protein family. By making aspecific mutation in TNF-gamma in such residues as tyrosine-15 (asnumbered in SEQ IN NO:2), leucine-35, glycine-41, tyrosine-43,tyrosine-46, glutamine-48, leucine-90, leucine-116, glycine-119,aspartic acid-120, phenylalanine-141, phenylalanine-142, andleucine-147, it is likely that an noticeable effect on biologicalactivity will be observed. These identical amino acid residues are, ofcourse, present in the corresponding positions of TNF-gamma-beta shownin SEQ ID NO:20.

The present invention also encompasses fragments of the above-describedTNF-gamma polypeptides. Polypeptide fragments of the present inventioninclude polypeptides comprising an amino acid sequence contained in SEQID NO:2, encoded by the cDNA contained in the deposited clone (HUVEO91),or encoded by nucleic acids which hybridize (e.g. under stringenthybridization conditions) to the nucleotide sequence contained in thedeposited clones, that shown in FIGS. 1A-C (SEQ ID NO:1) and/or FIGS.20A and 20B (SEQ ID NO:19), or the complementary strand thereto.

Polypeptide fragments may be “free-standing” or comprised within alarger polypeptide of which the fragment forms a part or region, mostpreferably as a single continuous region. Representative examples ofpolypeptide fragments of the invention, included, for example, fragmentsthat comprise or alternatively, consist of, from about amino acidresidues, 1 to 20, 21 to 40, 41 to 60, 61 to 83, 84 to 100, 101 to 120,121 to 140, 141 to 160, 160 to 167, 161 to 174, 161 to 180, 181 to 200,201 to 220, 221 to 240, 241 to 251 of SEQ ID NO:2 and/or SEQ ID NO:20.Moreover, polypeptide fragments can be at least about 20, 30, 40, 50,60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 amino acids in length. Inthis context “about” includes the particularly recited ranges, larger orsmaller by several (i.e. 5, 4, 3, 2 or 1) amino acids, at either extremeor at both extremes.

In other embodiments, the fragments or polypeptides of the invention(i.e., those described herein) are not larger than 250, 225, 200, 185,175, 170, 165, 160, 155, 150, 145, 140, 135, 130, 125, 120, 115, 110,105, 100, 90, 80, 75, 60, 50, 40, 30 or 25 amino acids residues inlength.

Further preferred embodiments encompass polypeptide fragmentscomprising, or alternatively consisting of, the mature domain ofTNF-gamma-alpha (amino acid residues 1-147 of SEQ ID NO:2), theintracellular domain of TNF-gamma-beta (amino acid residues 1-35 of SEQID NO:20), the transmembrane domain of TNF-gamma-beta (amino acidresidues 36-61 of SEQ ID NO:20), and/or the extracellular domain ofTNF-gamma-beta (amino acid residues 62-251 of SEQ ID NO:20).

In specific embodiments, polypeptide fragments of the inventioncomprise, or alternatively, consist of, amino acid residues leucine-35to valine-49, tryptophan-104 to leucine-116, glycine-119 to serine-127,lysine-139 to leucine-147 of SEQ ID NO:2). These domains are regions ofhigh identity identified by comparison of the TNF family memberpolypeptides shown in FIGS. 2A, 2B, and 2C.

Among the especially preferred fragments of the invention are fragmentscharacterized by structural or functional attributes of TNF-gamma. Suchfragments include amino acid residues that comprise alpha-helix andalpha-helix forming regions (“alpha-regions”), beta-sheet andbeta-sheet-forming regions (“beta-regions”), turn and turn-formingregions (“turn-regions”), coil and coil-forming regions(“coil-regions”), hydrophillic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, surface forming regions,and high antigenic index regions (i.e., regions of polypeptidesconsisting of amino acid residues having an antigenic index of or equalto greater than 1.5, as identified using the default parameters of theJameson-Wolf program) of TNF-gamma. Certain preferred regions are thosedisclosed in FIG. 17 and include, but are not limited to, regions of theaforementioned types identified by analysis of the amino acid sequencedepicted in FIGS. 1A-C, such preferred regions include; Garnier-Robsonpredicted alpha-regions, beta-regions, turn-regions, and coil-regions;Chou-Fasman predicted alpha-regions, beta-regions, turn-regions, andcoil-regions; Kyte-Doolittle predicted hydrophilic and hydrophobicregions; Eisenberg alpha and beta amphipathic regions; Eminisurface-forming regions; and Jameson-Wolf high antigenic index regions,as predicted using the default parameters of these computer programs.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

Additionally, analogs of the invention include a proprotein which can beactivated by cleavage of the proprotein portion to produce an activemature polypeptide.

In another embodiment, the invention provides a TNF-gamma polypeptide(e.g., fragment) comprising, or alternatively, consisting of, anepitope-bearing portion of a polypeptide of the invention. The epitopeof this polypeptide portion is an immunogenic or antigenic epitope of apolypeptide of the invention. An “immunogenic epitope” is defined as apart of a protein that elicits an antibody response when the wholeprotein is the immunogen. On the other hand, a region of a proteinmolecule to which an antibody can bind is defined as an “antigenicepitope”. The number of immunogenic epitopes of a protein generally isless than the number of antigenic epitopes (see, for instance, Geysen,et al., Proc. Natl. Acad. Sci. USA 81:3998-4002; (1983)).

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein (see, for instance, Sutcliffe, J. G., et aL, Science219:660-666 (1983)). Peptides capable of eliciting protein-reactive seraare frequently represented in the primary sequence of a protein, can becharacterized by a set of simple chemical rules, and are confinedneither to immunodominant regions of intact proteins (i.e., immunogenicepitopes) nor to the amino or carboxyl termini. Antigenicepitope-bearing peptides and polypeptides of the invention are thereforeuseful to raise antibodies, including monoclonal antibodies, that bindspecifically to a polypeptide of the invention (see, for instance,Wilson, et aL, Cell 37:767-778 (1984)).

Antigenic epitope-bearing peptides and polypeptides of the inventionpreferably contain a sequence of at least seven, more preferably atleast nine and most preferably between about 15 to about 30 amino acidscontained within the amino acid sequence of a polypeptide of theinvention. Non-limiting examples of antigenic polypeptides or peptidesthat can be used to generate TNF-gamma-specific antibodies include: apolypeptide comprising amino acid residues from about Thr-24 to aboutAsn-32 in SEQ ID NO:2; a polypeptide comprising amino acid residues fromabout Ile-37 to about Ile-45 in SEQ ID NO:2; a polypeptide comprisingamino acid residues from about Met-54 to about Arg-62 in SEQ ID NO:2; apolypeptide comprising amino acid residues from about Gln-63 to aboutAsp-71 in SEQ ID NO:2; a polypeptide comprising amino acid residues fromabout Glu-57 to about Gly-65 in SEQ ID NO:2; a polypeptide comprisingamino acid residues from about Val-80 to about Thr-88 in SEQ ID NO:2; apolypeptide comprising amino acid residues from about Leu-116 to aboutVal-124 in SEQ ID NO:2; and a polypeptide comprising amino acid residuesfrom about Asp-133 to about Phe-141 in SEQ ID NO:2. These polypeptidefragments have been determined to bear antigenic epitopes of theTNF-gamma protein by the analysis of the Jameson-Wolf antigenic index,as shown in FIG. 17, above.

One of ordinary skill in the art may easily determine antigenic regionsfor TNF-gamma-beta by using data prepared through a DNA*STAR analysis ofthe TNF-gamma-beta polypeptide sequence (SEQ ID NO:20) using the defaultparameters and selecting regions with a high antigenic index asdescribed above.

In another aspect, the invention provides peptides and polypeptidescomprising epitope-bearing portions of the polypeptides of the presentinvention. These epitopes are immunogenic or antigenic epitopes of thepolypeptides of the present invention. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response in vivowhen the whole polypeptide of the present invention, or fragmentthereof, is the immunogen. On the other hand, a region of a polypeptideto which an antibody can bind is defined as an “antigenic determinant”or “antigenic epitope.” The number of in vivo immunogenic epitopes of aprotein generally is less than the number of antigenic epitopes. See,e.g., Geysen, et al. (1983) Proc. Natl. Acad. Sci. USA 81:3998-4002.However, antibodies can be made to any antigenic epitope, regardless ofwhether it is an immunogenic epitope, by using methods such as phagedisplay. See e.g., Petersen G. et al. (1995) Mol. Gen. Genet.249:425-431. Therefore, included in the present invention are bothimmunogenic epitopes and antigenic epitopes.

A list of exemplified amino acid sequences comprising immunogenicepitopes is described above. It is pointed out that the list ofimmunogenic epitopes only lists amino acid residues comprising epitopespredicted to have the highest degree of antigenicity using the algorithmof Jameson and Wolf, (1988) Comp. Appl. Biosci. 4:181-186 (saidreferences incorporated by reference in their entireties). TheJameson-Wolf antigenic analysis was performed using the computer programPROTEAN, using default parameters (Version 3.11 for the Power MacIntosh,DNASTAR, Inc., 1228 South Park Street Madison, Wis.). Portions ofpolypeptides not listed in the above list of immunogenic epitopes arenot considered non-immunogenic. The immunogenic epitopes listed above isan exemplified list, not an exhaustive list, because other immunogenicepitopes are merely not recognized as such by the particular algorithmused. Amino acid residues comprising other immunogenic epitopes may beroutinely determined using algorithms similar to the Jameson-Wolfanalysis or by in vivo testing for an antigenic response using methodsknown in the art. See, e.g., Geysen et al., supra; U.S. Pat. Nos.4,708,781; 5,194,392; 4,433,092; and 5,480,971 (said referencesincorporated by reference in their entireties).

It is particularly pointed out that the amino acid sequences listedabove comprise immunogenic epitopes. The list of immunogenic epitopeslists only the critical residues of immunogenic epitopes determined bythe Jameson-Wolf analysis. Thus, additional flanking residues on eitherthe N-terminal, C-terminal, or both N- and C-terminal ends may be addedto the sequences listed above to generate an epitope-bearing polypeptideof the present invention. Therefore, the immunogenic epitopes listedabove may include additional N-terminal or C-terminal amino acidresidues. The additional flanking amino acid residues may be contiguousflanking N-terminal and/or C-terminal sequences from the polypeptides ofthe present invention, heterologous polypeptide sequences, or mayinclude both contiguous flanking sequences from the polypeptides of thepresent invention and heterologous polypeptide sequences.

Polypeptides of the present invention comprising immunogenic orantigenic epitopes are at least 7 amino acids residues in length. “Atleast” means that a polypeptide of the present invention comprising animmunogenic or antigenic epitope may be 7 amino acid residues in lengthor any integer between 7 amino acids and the number of amino acidresidues of the full length polypeptides of the invention. Preferredpolypeptides comprising immunogenic or antigenic epitopes are at least10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,or 100 amino acid residues in length. However, it is pointed out thateach and every integer between 7 and the number of amino acid residuesof the full length polypeptide are included in the present invention.

The immuno and antigenic epitope-bearing fragments may be specified byeither the number of contiguous amino acid residues, as described above,or further specified by N-terminal and C-terminal positions of thesefragments on the amino acid sequence of SEQ ID NO:2. Every combinationof a N-terminal and C-terminal position that a fragment of, for example,at least 7 or at least 15 contiguous amino acid residues in length couldoccupy on the amino acid sequence of SEQ ID NO:2 is included in theinvention. Again, “at least 7 contiguous amino acid residues in length”means 7 amino acid residues in length or any integer between 7 aminoacids and the number of amino acid residues of the full lengthpolypeptide of the present invention. Specifically, each and everyinteger between 7 and the number of amino acid residues of the fulllength polypeptide are included in the present invention. Further,immuno- and antigenic epitope-bearing fragments may be specified in thesame way for TNF-gamma-beta by using the techniques described herein.

Immunogenic and antigenic epitope-bearing polypeptides of the inventionare useful, for example, to make antibodies which specifically bind thepolypeptides of the invention, and in immunoassays to detect thepolypeptides of the present invention. The antibodies are useful, forexample, in affinity purification of the polypeptides of the presentinvention. The antibodies may also routinely be used in a variety ofqualitative or quantitative immunoassays, specifically for thepolypeptides of the present invention using methods known in the art.See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press; 2nd Ed. 1988).

The epitope-bearing polypeptides of the present invention may beproduced by any conventional means for making polypeptides includingsynthetic and recombinant methods known in the art. For instance,epitope-bearing peptides may be synthesized using known methods ofchemical synthesis. For instance, Houghten has described a simple methodfor the synthesis of large numbers of peptides, such as 10-20 mgs of 248individual and distinct 13 residue peptides representing single aminoacid variants of a segment of the HA1 polypeptide, all of which wereprepared and characterized (by ELISA-type binding studies) in less thanfour weeks (Houghten, R. A. Proc. Natl. Acad. Sci. USA 82:5131-5135(1985)). This “Simultaneous Multiple Peptide Synthesis (SMPS)” processis further described in U.S. Pat. No. 4,631,211 to Houghten andcoworkers (1986). In this procedure the individual resins for thesolid-phase synthesis of various peptides are contained in separatesolvent-permeable packets, enabling the optimal use of the manyidentical repetitive steps involved in solid-phase methods. A completelymanual procedure allows 500-1000 or more syntheses to be conductedsimultaneously (Houghten et al. (1985) Proc. Natl. Acad. Sci.82:5131-5135 at 5134.

Epitope-bearing polypeptides of the present invention are used to induceantibodies according to methods well known in the art including, but notlimited to, in vivo immunization, in vitro immunization, and phagedisplay methods. See, e.g., Sutcliffe, et al., supra; Wilson, et al.,supra, and Bittle, et al. (1985) J. Gen. Virol. 66:2347-2354. If in vivoimmunization is used, animals may be immunized with free peptide;however, anti-peptide antibody titer may be boosted by coupling of thepeptide to a macromolecular carrier, such as keyhole limpet hemacyanin(KLH) or tetanus toxoid. For instance, peptides containing cysteineresidues may be coupled to a carrier using a linker such as-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde. Animals such as rabbits, rats and mice are immunizedwith either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μgs of peptide or carrier protein and Freund's adjuvant.Several booster injections may be needed, for instance, at intervals ofabout two weeks, to provide a useful titer of anti-peptide antibodywhich can be detected, for example, by ELISA assay using free peptideadsorbed to a solid surface. The titer of anti-peptide antibodies inserum from an immunized animal may be increased by selection ofanti-peptide antibodies, for instance, by adsorption to the peptide on asolid support and elution of the selected antibodies according tomethods well known in the art.

As one of skill in the art will appreciate, and discussed above, thepolypeptides of the present invention comprising an immunogenic orantigenic epitope can be fused to heterologous polypeptide sequences.For example, the polypeptides of the present invention may be fused withthe constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portionsthereof (CH1, CH2, CH3, any combination thereof including both entiredomains and portions thereof) resulting in chimeric polypeptides. Thesefusion proteins facilitate purification, and show an increased half-lifein vivo. This has been shown, e.g., for chimeric proteins consisting ofthe first two domains of the human CD4-polypeptide and various domainsof the constant regions of the heavy or light chains of mammalianimmunoglobulins. See, e.g., EPA 0,394,827; Traunecker et al. (1988)Nature 331:84-86. Fusion proteins that have a disulfide-linked dimericstructure due to the IgG portion can also be more efficient in bindingand neutralizing other molecules than monomeric polypeptides orfragments thereof alone. See, e.g., Fountoulakis et al. (1995) J.Biochem. 270:3958-3964. Nucleic acids encoding the above epitopes canalso be recombined with a gene of interest as an epitope tag to aid indetection and purification of the expressed polypeptide.

The epitope-bearing peptides and polypeptides of the produced by anyconventional means (see, for example, Houghten, R. A., et aL, Proc.Natl. Acad Sci. USA 82:5131-5135 (1985); and U.S. Pat. No. 4,631,211 toHoughten, et al. (1986)).

Epitope-bearing peptides and polypeptides of the invention inventionhave uses which include, but are not limited to, inducing antibodiesaccording to methods well known in the art (see, for instance,Sutcliffe, et aL, supra; Wilson, et aL, supra; Chow, M., et al., Proc.NatL Acad Sci. USA 82:910-914; and Bittle, F. J., et aL, J Gen. ViroL66:2347-2354 (1985)). Immunogenic epitope-bearing peptides of theinvention, i.e., those parts of a protein that elicit an antibodyresponse when the whole protein is the immunogen, are identifiedaccording to methods known in the art (see, for instance, Geysen, et aL,supra). Further still, U.S. Pat. No. 5,194,392, issued to Geysen,describes a general method of detecting or determining the sequence ofmonomers (amino acids or other compounds) which is a topologicalequivalent of the epitope (i.e., a “mimotope”) which is complementary toa particular paratope (antigen binding site) of an antibody of interest.More generally, U.S. Pat. No. 4,433,092, issued to Geysen, describes amethod of detecting or determining a sequence of monomers which is atopographical equivalent of a ligand which is complementary to theligand binding site of a particular receptor of interest (e.g. DR3).Similarly, U.S. Pat. No. 5,480,971, issued to Houghten and colleagues,on Peralkylated Oligopeptide Mixtures discloses linear C1-C7-alkylperalkylated oligopeptides and sets and libraries of such peptides, aswell as methods for using such oligopeptide sets and libraries fordetermining the sequence of a peralkylated oligopeptide thatpreferentially binds to an acceptor molecule of interest. Thus,non-peptide analogs of the epitope-bearing peptides of the inventionalso can be made routinely by these methods.

As one of skill in the art will appreciate, TNF-gamma-alpha and/orTNF-gamma-beta polypeptides of the present invention and theepitope-bearing fragments thereof described above can be combined withparts of the constant domain of immunoglobulins (IgG), resulting inchimeric polypeptides. These fusion proteins facilitate purification andshow an increased half-life in vivo. This has been shown, e.g., forchimeric proteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins (EP A 394,827: Traunecker,et al., Nature 331:84-86 (1988)). Fusion proteins that have adisulfide-linked dimeric structure due to the IgG part can also be moreefficient in binding and neutralizing other molecules than the monomericTNF-gamma protein or protein fragment alone (Fountoulakis, et al., J.Biochem. 270:3958-3964 (1995)). As an example, one such TNF-gamma-Fcfusion has been produced herein as described above.

Fragments (i.e., portions) of the TNF-gamma polypeptides of the presenton have uses which include, but are not limited to, intermediates forproducing full-length polypeptides.

For many proteins, including the extracellular domain of a membraneassociated protein or the mature form(s) of a secreted protein, it isknown in the art that one or more amino acids may be deleted from theN-terminus or C-terminus without substantial loss of biologicalfunction. For instance, Ron and colleagues (J. Biol. Chem.,268:2984-2988 (1993)) reported modified KGF proteins that had heparinbinding activity even if 3, 8, or 27 N-terminal amino acid residues weremissing. Further, several investigators have reported TNF-a muteins inwhich two, four or seven N-terminal amino acids had been removed whichshowed a 2- to 3-fold increase in functional activity when compared tothe naturally-occurring TNF-a polypeptide (Creasey, A. A., et al.,Cancer Res. 47:145-149 (1987); Sidhu, R. S. and Bollon, A. P. AnticancerRes. 9:1569-1576 (1989); Kamijo, R., et al., Biochem. Biophys. Res.Comm. 160:820-827 (1989)). Further, even if deletion of one or moreamino acids from the N-terminus or C-terminus of a protein results inmodification or loss of one or more biological functions of the protein,other TNF-gamma functional activities may still be retained

In the present case, since the proteins of the invention are members ofthe TNF polypeptide family, deletions of N-terminal amino acids up tothe leucine residue at position 35 of SEQ ID NO:2 (which correspondsexactly to the leucine residue at position 134 of SEQ ID NO:20) mayretain some biological activity such as regulation of growth anddifferentiation of many types of hematopoietic and endothelial cells.Polypeptides having further N-terminal deletions including theleucine-36 residue in SEQ ID NO:2 (corresponding to leucine-135 in SEQID NO:20) would not be expected to retain such biological activitiesbecause it is known that this residue in TNF-related polypeptides is inthe beginning of the conserved domain required for biologicalactivities.

However, even if deletion of one or more amino acids from the N-terminusof a full-length TNP-gamma polypeptide results in modification or lossof one or more biological functions of the polypeptide, other biologicalactivities may still be retained. Thus, the ability of the shortenedpolypeptide to induce and/or bind to antibodies which recognize thefull-length or mature form of the polypeptide generally will be retainedwhen less than the majority of the residues of the full-length or maturepolypeptide are removed from the N-terminus. Whether a particularpolypeptide lacking N-terminal residues of a complete polypeptideretains such immunologic activities can readily be determined by routinemethods described herein and otherwise known in the art.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the amino acidsequence of the TNF-gamma-alpha shown in SEQ ID NO:2, up to the leucineresidue at position number 35, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues n¹-149 of SEQ ID NO:2,where n¹ is an integer in the range of −27 to 35, and 35 is the positionof the first residue from the N-terminus of the complete TNF-gammapolypeptide (shown in SEQ ID NO:2) believed to be required forregulation of growth and differentiation of many types of hematopoieticand endothelial cells.

In specific embodiments, the invention provides polynucleotides encodingpolypeptides comprising, or alternatively, consisting of, the amino acidsequence of residues: −27 to 147, −26 to 147, −25 to 147, −24 to 147,−23 to 147, −22 to 147, −21 to 147, −20 to 147, −19 to 147, −18 to 147,−17 to 147, −16 to 147, −15 to 147, −14 to 147, −13 to 147, −12 to 147,−11 to 147, −10 to 147, −9 to 147, −8 to 147, −7 to 147, −6 to 147, −5to 147, 4 to 147, −3 to 147, −2 to 147, −1 to 147, 1 to 147, 2 to 147, 3to 147, 4 to 147, 5 to 147, 6 to 147, 7 to 147, 8 to 147, 9 to 147, 10to 147, 11 to 147, 12 to 147, 13 to 147, 14 to 147, 15 to 147, 16 to147, 17 to 147, 18 to 147, 19 to 147, 20 to 147, 21 to 147, 22 to 147,23 to 147, 24 to 147, 27 to 147, 26 to 147, 27 to 147, 28 to 147, 29 to147, 30 to 147, 31 to 147, 32 to 147, 33 to 147, 34 to 147, and 35 to147 of SEQ ID NO:2. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the amino acidsequence of the TNF-gamma-beta shown in SEQ ID NO:20, up to the leucineresidue at position number 134, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues n²-251 of SEQ ID NO:20,where n² is an integer in the range of 1 to 134, and 135 is the positionof the first residue from the N-terminus of the complete TNF-gamma-betapolypeptide (shown in SEQ ID NO:20) believed to be required forregulation of growth and differentiation of many types of hematopoieticand endothelial cells activity of the TNF-gamma-beta polypeptide.

In specific embodiments, the invention provides polynucleotides encodingpolypeptides comprising, or alternatively, consisting of, the amino acidsequence of residues: 1 to 251, 2 to 251, 3 to 251, 4 to 251, 5 to 251,6 to 251, 7 to 251, 8 to 251, 9 to 251, 10 to 251, 11 to 251, 12 to 251,13 to 251, 14 to 251, 15 to 251, 16 to 251, 17 to 251, 18 to 251, 19 to251, 20 to 251, 21 to 251, 22 to 251, 23 to 251, 24 to 251, 25 to 251,26 to 251, 27 to 251, 28 to 251, 29 to 251, 30 to 251, 31 to 251, 32 to251, 33 to 251, 34 to 251, 35 to 251, 36 to 251, 37 to 251, 38 to 251,39 to 251, 40 to 251, 41 to 251, 41 to 251, 42 to 251, 43 to 251, 44 to251, 45 to 251, 46 to 251, 47 to 251, 48 to 251, 49 to 251, 50 to 251,51 to 251, 52 to 251, 53 to 251, 54 to 251, 55 to 251, 56 to 251, 57 to251, 58 to 251, 59 to 251, 60 to 251, 61 to 251, 62 to 251, 63 to 251,64 to 251, 65 to 251, 66 to 251, 67 to 251, 68 to 251, 69 to 251, 70 to251, 71 to 251, 72 to 251, 73 to 251, 74 to 251, 75 to 251, 76 to 251,77 to 251, 78 to 251, 79 to 251, 80 to 251, 81 to 251, 82 to 251, 83 to251, 84 to 251, 85 to 251, 86 to 251, 87 to 251, 88 to 251, 89 to 251,90 to 251, 91 to 251, 92 to 251, 93 to 251, 94 to 251, 95 to 251, 96 to251, 97 to 251, 98 to 251, 99 to 251, 100 to 251, 101 to 251, 102 to251, 103 to 251, 104 to 251, 105 to 251, 106 to 251, 107 to 251, 108 to251, 109 to 251, 110 to 251, 111 to 251, 112 to 251, 113 to 251, 114 to251, 115 to 251, 116 to 251, 117 to 251, 118 to 251, 119 to 251, 120 to251, 121 to 251, 122 to 251, 123 to 251, 124 to 251, 125 to 251, 126 to251, 127 to 251, 128 to 251, 129 to 251, 130 to 251, 131 to 251, 133 to251, 134 to 251, and 134 to 251 of SEQ ID NO:20. Polynucleotidesencoding these polypeptides are also encompassed by the invention.

Several amino acids of the TNF-gamma polypeptide are highly conservedacross the known members of the TNF-related protein family. By making aspecific mutation in TNF-gamma in such residues as tryptophan-15 (asnumbered in SEQ ID NO:2), leucine-35, glycine-41, tyrosine-43,tyrosine-46, glutamine-48, leucine-90, leucine-116, glycine-119,aspartic acid-120, phenylalanine-141, phenylalanine-142, andleucine-147, it is likely that an noticeable effect on biologicalactivity will be observed. These identical amino acid residues are, ofcourse, present in the corresponding positions of TNF-gamma-beta shownin SEQ ID NO:20.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the predictedmature amino acid sequence of the TNF-gamma-alpha shown in FIGS. 1A-C(SEQ ID NO:2), up to the phenylalanine residue at position number 169 ofthe sequence shown in FIGS. 1A-C (which corresponds to position number142 of SEQ ID NO:2) and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides comprising theamino acid sequence of residues n³-174 of the sequence shown in FIGS.1A-C (n³-147 of SEQ ID NO:2), where n³ is an integer in the range of 1to 169, and 170 is the position of the first residue from the N-terminusof the complete TNF-gamma-alpha polypeptide believed to be required forat least immunogenic activity of the TNF-gamma-alpha polypeptide.

More in particular, the invention provides polynucleotides encodingpolypeptides comprising, or alternatively consisting of, the amino acidsequence of residues of R-2 to L-174; R-3 to L-174; F-4 to L-174; L-5 toL-174; S-6 to L-174; K-7 to L-174; V-8 to L-174; Y-9 to L-174; S-10 toL-174; F-11 to L-174; P-12 to L-174; M-13 to L-174; R-14 to L-174; K-15to L-174; L-16 to L-174; I-17 to L-174; L-18 to L-174; F-19 to L-174;L-20 to L-174; V-21 to L-174; F-22 to L-174; P-23 to L-174; V-24 toL-174; V-25 to L-174; R-26 to L-174; Q-27 to L-174; T-28 to L-174; P-29to L-174; T-30 to L-174; Q-31 to L-174; H-32 to L-174; F-33 to L-174;K-34 to L-174; N-35 to L-174; Q-36 to L-174; F-37 to L-174; P-38 toL-174; A-39 to L-174; L-40 to L-174; H-41 to L-174; W-42 to L-174; E-43to L-174; H-44 to L-174; E-45 to L-174; L-46 to L-174; G-47 to L-174;L-48 to L-174: A-49 to L-174; F-50 to L-174; T-51 to L-174; K-52 toL-174; N-53 to L-174; R-54 to L-174; M-55 to L-174; N-56 to L-174; Y-57to L-174; T-58 to L-174; N-59 to L-174; K-60 to L-174; F-61 to L-174;L-62 to L-174; L-63 to L-174; I-64 to L-174; P-65 to L-174; E-66 toL-174; S-67 to L-174; G-68 to L-174; D-69 to L-174; Y-70 to L-174; F-71to L-174; I-72 to L-174; Y-73 to L-174; S-74 to L-174; Q-75 to L-174;V-76 to L-174; T-77 to L-174; F-78 to L-174; R-79 to L-174; G-80 toL-174; M-81 to L-174; T-82 to L-174; S-83 to L-174; E-84 to L-174; C-85to L-174; S-86 to L-174; E-87 to L-174; I-88 to L-174; R-89 to L-174;Q-90 to L-174; A-91 to L-174; G-92 to L-174; R-93 to L-174; P-94 toL-174; N-95 to L-174; K-96 to L-174; P-97 to L-174; D-98 to L-174; S-99to L-174; I-100 to L-174; T-101 to L-174; V-102 to L-174; V-103 toL-174; I-104 to L-174; T-105 to L-174; K-106 to L-174; V-107 to L-174;T-108 to L-174; D-109 to L-174; S-110 to L-174; Y-111 to L-174; P-112 toL-174; E-113 to L-174; P-114 to L-174; T-115 to L-174; Q-116 to L-174;L-117 to L-174; L-118 to L-174; M-119 to L-174; G-120 to L-174; T-121 toL-174; K-122 to L-174; S-123 to L-174; V-124 to L-174; C-125 to L-174;E-126 to L-174; V-127 to L-174; G-128 to L-174; S-129 to L-174; N-130 toL-174; W-131 to L-174; F-132 to L-174; Q-133 to L-174; P-134 to L-174;I-135 to L-174; Y-136 to L-174; L-137 to L-174; G-138 to L-174; A-139 toL-174; M-140 to L-174; F-141 to L-174; S-142 to L-174; L-143 to L-174;Q-144 to L-174; E-145 to L-174; G-146 to L-174; D-147 to L-174; K-148 toL-174; L-149 to L-174; M-150 to L-174; V-151 to L-174; N-152 to L-174;V-153 to L-174; S-154 to L-174; D-155 to L-174; I-156 to L-174; S-157 toL-174; L-158 to L-174; V-159 to L-174; D-160 to L-174; Y-161 to L-174;T-162 to L-174; K-163 to L-174; E-164 to L-174; D-165 to L-174; K-166 toL-174; T-167 to L-174; F-168 to L-174; and F-169 to L-174 of theTNF-gamma-alpha sequence shown in FIGS. 1A-C (the TNF-gamma-alpha aminoacid sequence shown in FIGS. 1A-C is identical to that in SEQ ID NO:2,however, the numbering scheme differs between the two; the numbering ofthe above amino acid residues in this case reflects that of FIGS. 1A-C).Polynucleotides encoding these polypeptides are also encompassed by theinvention.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the predictedmature amino acid sequence of the TNF-gamma-beta shown in SEQ ID NO:20,up to the phenylalanine residue at position number 246 andpolynucleotides encoding such polypeptides. In particular, the presentinvention provides polypeptides comprising the amino acid sequence ofresidues n⁴-251 of SEQ ID NO:20, where n⁴ is an integer in the range of2 to 246, and 247 is the position of the first residue from theN-terminus of the complete TNF-gamma-beta polypeptide believed to berequired for at least immunogenic activity of the TNF-gamma-betaprotein.

More in particular, the invention provides polynucleotides encodingpolypeptides comprising, or alternatively consisting of, the amino acidsequence of residues of A-2 to L-251; E-3 to L-251; D-4 to L-251; L-5 toL-251; G-6 to L-251; L-7 to L-251; S-8 to L-251; F-9 to L-251; G-10 toL-251; E-11 to L-251; T-12 to L-251; A-13 to L-251; S-14 to L-251; V-15to L-251; E-16 to L-251; M-17 to L-251; L-18 to L-251; P-19 to L-251;E-20 to L-251; H-21 to L-251; G-22 to L-251; S-23 to L-251; C-24 toL-251; R-25 to L-251; P-26 to L-251; K-27 to L-251; A-28 to L-251; R-29to L-251; S-30 to L-251; S-31 to L-251; S-32 to L-251; A-33 to L-251;R-34 to L-251; W-35 to L-251; A-36 to L-251; L-37 to L-251; T-38 toL-251; C-39 to L-251; C-40 to L-251; L-41 to L-251; V-42 to L-251; L-43to L-251; L-44 to L-251; P-45 to L-251; F-46 to L-251; L-47 to L-251;A-49 to L-251; G-49 to L-251; L-50 to L-251; T-51 to L-251; T-52 toL-251; Y-53 to L-251; L-54 to L-251; L-55 to L-251; V-56 to L-251; S-57to L-251; Q-58 to L-251; L-59 to L-251; R-60 to L-251; A-61 to L-251;Q-62 to L-251; G-63 to L-251; E-64 to L-251; A-65 to L-251; C-66 toL-251; V-67 to L-251; Q-68 to L-251; F-69 to L-251; Q-70 to L-251; A-71to L-251; L-72 to L-251; K-73 to L-251; G-74 to L-251; Q-75 to L-251;E-76 to L-251; F-77 to L-251; A-78 to L-251; P-79 to L-251; S-80 toL-251; H-81 to L-251; Q-82 to L-251; Q-83 to L-251; V-84 to L-251; Y-85to L-251; A-86 to L-251; P-87 to L-251; L-88 to L-251; R-89 to L-251;A-90 to L-251; D-91 to L-251; G-92 to L-251; D-93 to L-251; K-94 toL-251; P-95 to L-251; R-96 to L-251; A-97 to L-251; H-98 to L-251; L-99to L-251; T-100 to L-251; V-101 to L-251; V-102 to L-251; R-103 toL-251; Q-104 to L-251; T-105 to L-251; P-106 to L-251; T-107 to L-251;Q-108 to L-251; H-109 to L-251; F-110 to L-251; K-111 to L-251; N-112 toL-251; Q-113 to L-251; F-114 to L-251; P-115 to L-251; A-116 to L-251;L-117 to L-251; H-118 to L-251; W-119 to L-251; E-120 to L-251; H-121 toL-251; E-122 to L-251; L-123 to L-251; G-124 to L-251; L-125 to L-251:A-126 to L-251; F-127 to L-251; T-128 to L-251; K-129 to L-251; N-130 toL-251; R-131 to L-251; M-132 to L-251; N-133 to L-251; Y-134 to L-251;T-135 to L-251; N-136 to L-251; K-137 to L-251; F-138 to L-251; L-139 toL-251; L-140 to L-251; I-141 to L-251; P-142 to L-251; E-143 to L-251;S-144 to L-251; G-145 to L-251; D-146 to L-251; Y-147 to L-251; F-148 toL-251; I-149 to L-251; Y-150 to L-251; S-151 to L-251; Q-152 to L-251;V-153 to L-251; T-154 to L-251; F-155 to L-251; R-156 to L-251; G-157 toL-251; M-158 to L-251; T-159 to L-251; S-160 to L-251; E-161 to L-251;C-162 to L-251; S-163 to L-251; E-164 to L-251; I-165 to L-251; R-166 toL-251; Q-167 to L-251; A-168 to L-251; G-169 to L-251; R-170 to L-251;P-171 to L-251; N-172 to L-251; K-173 to L-251; P-174 to L-251; D-175 toL-251; S-176 to L-251; I-177 to L-251; T-178 to L-251; V-179 to L-251;V-180 to L-251; I-181 to L-251; T-182 to L-251; K-183 to L-251; V-184 toL-251; T-185 to L-251; D-186 to L-251; S-187 to L-251; Y-188 to L-251;P-189 to L-251; E-190 to L-251; P-191 to L-251; T-192 to L-251; Q-193 toL-251; L-194 to L-251; L-195 to L-251; M-196 to L-251; G-197 to L-251;T-198 to L-251; K-199 to L-251; S-200 to L-251; V-201 to L-251; C-202 toL-251; E-203 to L-251; V-204 to L-251; G-205 to L-251; S-206 to L-251;N-207 to L-251; W-208 to L-251; F-209 to L-251; Q-210 to L-251; P-211 toL-251; I-212 to L-251; Y-213 to L-251; L-214 to L-251; G-215 to L-251;A-216 to L-251; M-217 to L-251; F-218 to L-251; S-219 to L-251; L-220 toL-251; Q-221 to L-251; E-222 to L-251; G-223 to L-251; D-224 to L-251;K-225 to L-251; L-226 to L-251; M-227 to L-251; V-228 to L-251; N-229 toL-251; V-230 to L-251; S-231 to L-251; D-232 to L-251; I-233 to L-251;S-234 to L-251; L-235 to L-251; V-236 to L-251; D-237 to L-251; Y-238 toL-251; T-239 to L-251; K-240 to L-251; E-241 to L-251; D-242 to L-251;K-243 to L-251; T-244 to L-251; F-245 to L-251; and F-246 to L-251 ofthe TNF-gamma-beta sequence shown in SEQ ID NO:20. Polynucleotidesencoding these polypeptides are also encompassed by the invention.

Similarly, many examples of biologically functional C-terminal deletionmuteins are known. For instance, Interferon gamma shows up to ten timeshigher activities by deleting 8 to 10 amino acid residues from thecarboxy terminus of the protein (Dobeli, et al., J. Biotechnology7:199-216 (1988)). Further, several investigators have reportedbiologically inactive TNF-a muteins in which as few as two amino acidshad been removed from the C-terminus (Carlino, J. A., et al., J. Biol.Chem. 262:958-961 (1987); Creasey, A. A., et al., Cancer Res. 47:145-149(1987); Sidhu, R. S. and Bollon, A. P. Anticancer Res. 9:1569-1576(1989); Gase, K., et al., Immunology 71:368-371 (1990)).

In the present case, since the proteins of the invention are members ofthe TNF polypeptide family, deletions of C-terminal amino acids up tothe leucine at position 146 of SEQ ID NO:2 (which corresponds to theleucine at position 250 of SEQ ID NO:20) may retain some biologicalactivity such as regulation of growth and differentiation of many typesof hematopoietic and endothelial cells. Polypeptides having furtherC-terminal deletions including the leucine residue at position 146 ofSEQ ID NO:2 (or the leucine residue at position 250 of SEQ ID NO:20)would not be expected to retain such biological activities because it isknown that this residue in TNF-related polypeptides is in the beginningof the conserved domain required for biological activities.

However, even if deletion of one or more amino acids from the C-terminusof a protein results in modification of loss of one or more biologicalfunctions of the protein, other biological activities may still beretained. Thus, the ability of the shortened protein to induce and/orbind to antibodies which recognize the complete or mature form of theprotein generally will be retained when less than the majority of theresidues of the complete or mature protein are removed from theC-terminus. Whether a particular polypeptide lacking C-terminal residuesof a complete protein retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art.

In additional embodiments, the present invention further providespolypeptides having one or more residues removed from the carboxyterminus of the amino acid sequence of the TNF-gamma-alpha shown in SEQID NO:2, up to the leucine residue at position 146 of SEQ ID NO:2, andpolynucleotides encoding such polypeptides. In particular, the presentinvention provides polypeptides having the amino acid sequence ofresidues −27-m¹ of the amino acid sequence in SEQ ID NO:2, where m¹ isany integer in the range of 146 to 147, and residue 146 is the positionof the first residue from the C-terminus of the complete TNF-gamma-alphapolypeptide (shown in SEQ ID NO:2) believed to be required forregulation of growth and differentiation of many types of hematopoieticand endothelial cells by the TNF-gamma-alpha polypeptide.

More in particular, the invention provides polynucleotides encodingpolypeptides having the amino acid sequence of residues −27-146 and−27-147 of SEQ ID NO:2. Polynucleotides encoding these polypeptides alsoare provided.

The present invention also provides polypeptides having one or moreresidues removed from the carboxy terminus of the amino acid sequence ofthe TNF-gamma-beta shown in SEQ ID NO:20, up to the leucine residue atposition 250 of SEQ ID NO:20, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptideshaving the amino acid sequence of residues 1-m² of the amino acidsequence in SEQ ID NO:20, where m² is any integer in the range of 250 to251, and residue 249 is the position of the first residue from theC-terminus of the complete TNF-gamma-beta polypeptide (shown in SEQ IDNO:20) believed to be required for regulation of growth anddifferentiation of many types of hematopoietic and endothelial cells.

More in particular, the invention provides polynucleotides encodingpolypeptides having the amino acid sequence of residues 1-250 and 1-251of SEQ ID NO:20. Polynucleotides encoding these polypeptides also areprovided.

The invention also provides polypeptide fragments comprising, oralternatively consisting of, one or more amino acids deleted from boththe amino and the carboxyl termini of TNF-gamma-alpha, which may bedescribed generally as having residues n¹-m¹ of SEQ ID NO:2, where n andm are integers as described above. The invention further providespolypeptides having one or more amino acids deleted from both the aminoand the carboxyl termini of TNF-gamma-beta, which may be describedgenerally as having residues n²-m² of SEQ ID NO:20, where n² and m² areintegers as described above.

As mentioned above, even if deletion of one or more amino acids from theC-terminus of a polypeptide results in modification of loss of one ormore biological functions of the polypeptide, other biologicalactivities may still be retained. Thus, the ability of the shortenedTNF-gamma-alpha mutein to induce and/or bind to antibodies whichrecognize the full-length or mature of the polypeptide generally will beretained when less than the majority of the residues of the complete ormature polypeptide are removed from the C-terminus. Whether a particularpolypeptide lacking C-terminal residues of a full-length polypeptideretains such immunologic activities can readily be determined by routinemethods described herein and otherwise known in the art. It is notunlikely that a TNF-gamma-alpha mutein with a large number of deletedC-terminal amino acid residues may retain some biological or immungenicactivities.

In fact, peptides composed of as few as six TNF-gamma-alpha amino acidresidues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the TNF-gamma-alpha shown in FIGS. 1A-C (or in SEQ ID NO:2),up to the serine residue at position number 6 in FIGS. 1A-C (or −22 inSEQ ID NO:2), and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides comprising theamino acid sequence of residues 1-m³ of SEQ ID NO:2, where m³ is aninteger in the range of 6 to 174, and 6 is the position of the firstresidue from the C-terminus of the complete TNF-gamma-alpha polypeptidebelieved to be required for at least immunogenic activity ofTNF-gamma-alpha.

More in particular, the invention provides polynucleotides encodingpolypeptides comprising, or alternatively consisting of, the amino acidsequence of residues M-1 to L-173; M-1 to F-172; M-1 to A-171; M-1 toG-170; M-1 to F-169; M-1 to F-168; M-1 to T-167; M-1 to K-166; M-1 toD-165; M-1 to E-164; M-1 to K-163; M-1 to T-162; M-1 to Y-161; M-1 toD-160; M-1 to V-159; M-1 to L-158; M-1 to S-157; M-1 to I-156; M-1 toD-155; M-1 to S-154; M-1 to V-153; M-1 to N-152; M-1 to V-151; M-1 toM-150; M-1 to L-149; M-1 to K-148; M-1 to D-147; M-1 to G-146; M-1 toE-145; M-1 to Q-144; M-1 to L-143; M-1 to S-142; M-1 to F-141; M-1 toM-140; M-1 to A-139; M-1 to G-138; M-1 to L-137; M-1 to Y-136; M-1 toI-135; M-1 to P-134; M-1 to Q-133; M-1 to F-132; M-1 to W-131; M-1 toN-130; M-1 to S-129; M-1 to G-128; M-1 to V-127; M-1 to E-126; M-1 toC-125; M-1 to V-124; M-1 to S-123; M-1 to K-122; M-1 to T-121; M-1 toG-120; M-1 to M-119; M-1 to L-118; M-1 to L-117; M-1 to Q-116; M-1 toT-115; M-1 to P-114; M-1 to E-113; M-1 to P-112; M-1 to Y-111; M-1 toS-110; M-1 to D-109; M-1 to T-108; M-1 to V-107; M-1 to K-106; M-1 toT-105; M-1 to I-104; M-1 to V-103; M-1 to V-102; M-1 to T-101; M-1 toI-100; M-1 to S-99; M-1 to D-98; M-1 to P-97; M-1 to K-96; M-1 to N-95;M-1 to P-94; M-1 to R-93; M-1 to G-92; M-1 to A-91; M-1 to Q-90; M-1 toR-89; M-1 to I-88; M-1 to E-87; M-1 to S-86; M-1 to C-85; M-1 to E-84;M-1 to S-83; M-1 to T-82; M-1 to M-81; M-1 to G-80; M-1 to R-79; M-1 toF-78; M-1 to T-77; M-1 to V-76; M-1 to Q-75; M-1 to S-74; M-1 to Y-73;M-1 to I-72; M-1 to F-71; M-1 to Y-70; M-1 to D-69; M-1 to G-68; M-1 toS-67; M-1 to E-66; M-1 to P-65; M-1 to I-64; M-1 to L-63; M-1 to L-62;M-1 to F-61; M-1 to K-60; M-1 to N-59; M-1 to T-58; M-1 to Y-57; M-1 toN-56; M-1 to M-55; M-1 to R-54; M-1 to N-53; M-1 to K-52; M-1 to T-51;M-1 to F-50; M-1 to A-49; M-1 to L-48; M-1 to G-47; M-1 to L-46; M-1 toE-45; M-1 to H-44; M-1 to E-43; M-1 to W-42; M-1 to H-41; M-1 to L-40;M-1 to A-39; M-1 to P-38; M-1 to F-37; M-1 to Q-36; M-1 to N-35; M-1 toK-34; M-1 to F-33; M-1 to H-32; M-1 to Q-31; M-1 to T-30; M-1 to P-29;M-1 to T-28; M-1 to Q-27; M-1 to R-26; M-1 to V-25; M-1 to V-24; M-1 toP-23; M-1 to F-22; M-1 to V-21; M-1 to L-20; M-1 to F-19; M-1 to L-18;M-1 to I-17; M-1 to L-16; M-1 to K-15: M-1 to R-14; M-1 to M-13; M-1 toP-12; M-1 to F-11; M-1 to S-10; M-1 to Y-9; M-1 to V-8; M-1 to K-7; andM-1 to S-6 of the sequence of the TNF-gamma-alpha sequence shown inFIGS. 1A-C (the TNF-gamma-alpha amino acid sequence shown in FIGS. 1A-Cis identical to that in SEQ ID NO:2, however, the numbering schemediffers between the two; the numbering of the above amino acid residuesin this case reflects that of FIGS. 1A-C). Polynucleotides encodingthese polypeptides also are provided.

The invention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini of aTNF-gamma-alpha polypeptide, which may be described generally as havingresidues n³-m³ of SEQ ID NO:2, where n³ and m³ are integers as describedabove. Polynucleotides encoding the polypeptides are also encompassed bythe invention.

The present invention further provides polypeptides having one or moreresidues deleted from the carboxy terminus of the amino acid sequence ofthe TNF-gamma-beta shown in SEQ ID NO:20, up to the glycine residue atposition number 6, and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides comprising theamino acid sequence of residues 1-m⁴ of SEQ ID NO:20, where m⁴ is aninteger in the range of 6 to 250, and 6 is the position of the firstresidue from the C-terminus of the complete TNF-gamma-beta polypeptidebelieved to be required for at least immunogenic activity of theTNF-gamma-beta protein.

More in particular, the invention provides polynucleotides encodingpolypeptides comprising, or alternatively consisting of, the amino acidsequence of residues M-1 to L-250; M-1 to F-249; M-1 to A-248; M-1 toG-247; M-1 to F-246; M-1 to F-245; M-1 to T-244; M-1 to K-243; M-1 toD-242; M-1 to E-241; M-1 to K-240; M-1 to T-239; M-1 to Y-238; M-1 toD-237; M-1 to V-236; M-1 to L-235; M-1 to S-234; M-1 to I-233; M-1 toD-232; M-1 to S-231; M-1 to V-230; M-1 to N-229; M-1 to V-228; M-1 toM-227; M-1 to L-226; M-1 to K-225; M-1 to D-224; M-1 to G-223; M-1 toE-222; M-1 to Q-221; M-1 to L-220; M-1 to S-219; M-1 to F-218; M-1 toM-217; M-1 to A-216; M-1 to G-215; M-1 to L-214; M-1 to Y-213; M-1 toI-212; M-1 to P-211; M-1 to Q-210; M-1 to F-209; M-1 to W-208; M-1 toN-207; M-1 to S-206; M-1 to G-205; M-1 to V-204; M-1 to E-203; M-1 toC-202; M-1 to V-201; M-1 to S-200; M-1 to K-199; M-1 to T-198; M-1 toG-197; M-1 to M-196; M-1 to L-195; M-1 to L-194; M-1 to Q-193; M-1 toT-192; M-1 to P-191; M-1 to E-190; M-1 to P-189; M-1 to Y-188; M-1 toS-187; M-1 to D-186; M-1 to T-185; M-1 to V-184; M-1 to K-183; M-1 toT-182; M-1 to I-181; M-1 to V-180; M-1 to V-179; M-1 to T-178; M-1 toI-177; M-1 to S-176; M-1 to D-175; M-1 to P-174; M-1 to K-173; M-1 toN-172; M-1 to P-171; M-1 to R-170; M-1 to G-169; M-1 to A-168; M-1 toQ-167; M-1 to R-166; M-1 to I-165; M-1 to E-164; M-1 to S-163; M-1 toC-162; M-1 to E-161; M-1 to S-160; M-1 to T-159; M-1 to M-158; M-1 toG-157; M-1 to R-156; M-1 to F-155; M-1 to T-154; M-1 to V-153; M-1 toQ-152; M-1 to S-151; M-1 to Y-150; M-1 to I-149; M-1 to F-148; M-1 toY-147; M-1 to D-146; M-1 to G-145; M-1 to S-144; M-1 to E-143; M-1 toP-142; M-1 to I-141; M-1 to L-140; M-1 to L-139; M-1 to F-138; M-1 toK-137; M-1 to N-136; M-1 to T-135; M-1 to Y-134; M-1 to N-133; M-1 toM-132; M-1 to R-131; M-1 to N-130; M-1 to K-129; M-1 to T-128; M-1 toF-127; M-1 to A-126; M-1 to L-125; M-1 to G-124; M-1 to L-123; M-1 toE-122; M-1 to H-121; M-1 to E-120; M-1 to W-119; M-1 to H-118; M-1 toL-117; M-1 to A-116; M-1 to P-115; M-1 to F-114; M-1 to Q-113; M-1 toN-112; M-1 to K-111; M-1 to F-110; M-1 to H-109; M-1 to Q-108; M-1 toT-107; M-1 to P-106; M-1 to T-105; M-1 to Q-104; M-1 to R-103; M-1 toV-102; M-1 to V-101; M-1 to T-100; M-1 to L-99; M-1 to H-98; M-1 toA-97; M-1 to R-96; M-1 to P-95; M-1 to K-94; M-1 to D-93; M-1 to G-92;M-1 to D-91; M-1 to A-90; M-1 to R-89; M-1 to L-88; M-1 to P-87; M-1 toA-86; M-1 to Y-85; M-1 to V-84; M-1 to Q-83; M-1 to Q-82; M-1 to H-81;M-1 to S-80; M-1 to P-79; M-1 to A-78; M-1 to F-77; M-1 to E-76; M-1 toQ-75; M-1 to G-74; M-1 to K-73; M-1 to L-72; M-1 to A-71; M-1 to Q-70;M-1 to F-69; M-1 to Q-68; M-1 to V-67; M-1 to C-66; M-1 to A-65; M-1 toE-64; M-1 to G-63; M-1 to Q-62; M-1 to A-61; M-1 to R-60; M-1 to L-59;M-1 to Q-58; M-1 to S-57; M-1 to V-56; M-1 to L-55; M-1 to L-54; M-1 toY-53; M-1 to T-52; M-1 to T-51; M-1 to L-50; M-1 to G-49; M-1 to A-48;M-1 to L-47; M-1 to F-46; M-1 to P-45; M-1 to L-44; M-1 to L-43; M-1 toV-42; M-1 to L-41; M-1 to C-40; M-1 to C-39; M-1 to T-38; M-1 to L-37;M-1 to A-36; M-1 to W-35; M-1 to R-34; M-1 to A-33; M-1 to S-32; M-1 toS-31; M-1 to S-30; M-1 to R-29; M-1 to A-28; M-1 to K-27; M-1 to P-26;M-1 to R-25; M-1 to C-24; M-1 to S-23; M-1 to G-22; M-1 to H-21; M-1 toE-20; M-1 to P-19; M-1 to L-18; M-1 to M-17; M-1 to E-16; M-1 to V-15;M-1 to S-14; M-1 to A-13; M-1 to T-12; M-1 to E-11; M-1 to G-10; M-1 toF-9; M-1 to S-8; M-1 to L-7; and M-1 to G-6 of the sequence of theTNF-gamma-beta sequence shown in SEQ ID NO:20. Polynucleotides encodingthese polypeptides also are provided.

The invention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini of a TNF-gamma-betapolypeptide, which may be described generally as having residues n⁴-m⁴of SEQ ID NO:20, where n⁴ and m⁴ are integers as described above.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

Further embodiments of the invention are directed to polypeptidefragments comprising, or, alternatively, consisting of, amino acidsdescribed by the general formula m^(x) to n^(x), where m and ncorrespond to any one of the amino acid residues specified above forthese symbols, respectively, and x represents any integer.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

Specific embodiments of the invention are directed to nucleotidesequences encoding a polypeptide consisting of a portion of the completeTNF-gamma-alpha amino acid sequence encoded by the cDNA clone containedin ATCC Deposit No. 75927, where this portion excludes from 1 to about62 amino acids from the amino terminus of the complete amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 75927,or about 1 amino acid from the carboxy terminus, or any combination ofthe above amino terminal and carboxy terminal deletions, of the completeamino acid sequence encoded by the cDNA clone contained in ATCC DepositNo. 75927. Polynucleotides encoding all of the above deletion mutantpolypeptide forms also are provided.

In another embodiment, the invention is directed to a nucleotidesequence encoding a polypeptide consisting of a portion of the completeTNF-gamma-beta amino acid sequence encoded by the cDNA clone containedin ATCC Deposit No. 203055, where this portion excludes from 1 to about134 amino acids from the amino terminus of the complete amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 203055,or excludes a number of amino acids from the amino terminus of thecomplete amino acid sequence encoded by the cDNA clone contained in ATCCDeposit No. 203055 (where the number is selected from any integer from 1to 134), or about 1 amino acid from the carboxy terminus, or anycombination of the above amino terminal and carboxy terminal deletions,of the complete amino acid sequence encoded by the cDNA clone containedin ATCC Deposit No. 203055. Polynucleotides encoding all of the abovepolypeptides are also encompassed by the invention.

The invention further provides an isolated TNF-gamma polypeptidecomprising an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of the full-length TNF-gamma-alphapolypeptide having the complete amino acid sequence shown in SEQ ID NO:2(i.e., positions −27 to 147 of SEQ ID NO:2); (b) the amino acid sequenceof the full-length TNF-gamma-alpha polypeptide having the complete aminoacid sequence shown in SEQ ID NO:2 excepting the N-terminal methionine(i.e., positions −26 to 147 of SEQ ID NO:2); (c) the amino acid sequenceof the predicted mature TNF-gamma-alpha polypeptide having the aminoacid sequence at positions 1-147 in SEQ ID NO:2 (d) the complete aminoacid sequence encoded by the cDNA clone HUVEO91 contained in the ATCCDeposit No. 75927; (e) the complete amino acid sequence excepting theN-terminal methionine encoded by the cDNA clone contained in the ATCCDeposit No. 75927; and (f) the complete amino acid sequence of thepredicted mature TNF-gamma polypeptide encoded by the cDNA clone HUVEO91contained in the ATCC Deposit No. 75927. The polypeptides of the presentinvention also include polypeptides having an amino acid sequence atleast 70% identical, at least 80% identical, more to preferably at least90% identical, and still more preferably 95%, 96%, 97%, 98% or 99%identical to those described in (a), (b), (c), (d), (e) or (f), above,or fragments thereof, as described herein.

The invention further provides an isolated TNF-gamma polypeptidecomprising an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of the full-length TNF-gamma-betapolypeptide having the complete amino acid sequence shown in SEQ IDNO:20 (i.e., positions 1 to 251 of SEQ ID NO:20); (b) the amino acidsequence of the full-length TNF-gamma-beta polypeptide having thecomplete amino acid sequence shown in SEQ ID NO:20 excepting theN-terminal methionine (i.e., positions 2 to 251 of SEQ ID NO:20); (c)the amino acid sequence of the predicted mature TNF-gamma-betapolypeptide having the amino acid sequence at positions 62-251 in SEQ IDNO:20; (d) the complete amino acid sequence encoded by the cDNA cloneHEMCZ56 contained in the ATCC Deposit No. 203055; (e) the complete aminoacid sequence excepting the N-terminal methionine encoded by the cDNAclone HEMCZ56 contained in the ATCC Deposit No. 203055; and (f) thecomplete amino acid sequence of the predicted mature TNF-gammapolypeptide encoded by the cDNA clone HEMCZ56 contained in the ATCCDeposit No. 203055. The polypeptides of the present invention alsoinclude polypeptides having an amino acid sequence at least 70%identical, at least 80% identical, more preferably at least 90%identical, and still more preferably 95%, 96%, 97%, 98% or 99% identicalto those described in (a), (b), (c), (d), (e) or (f), above, orfragments thereof, as described herein. In specific embodiments, thesepolypeptides are at least 10 amino acids, at least 15 amino acids, atleast 20 amino acids, at least 25 amino acids, at least 30 amino acidsand more preferably at least 50 amino acids.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of a TNF-gammapolypeptide is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of the TNF-gamma polypeptide. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular polypeptide is at least90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the aminoacid sequence shown in FIGS. 1A-C (SEQ ID NO:2), the amino acid sequenceencoded by deposited cDNA clone HUVEO91, or fragments thereof, can bedetermined conventionally using known computer programs such the Bestfitprogram (Wisconsin Sequence Analysis Package, Version 8 for Unix,Genetics Computer Group, University Research Park, 575 Science Drive,Madison, Wis. 53711). When using Bestfit or any other sequence alignmentprogram to determine whether a particular sequence is, for instance, 95%identical to a reference sequence according to the present invention,the parameters are set, of course, such that the percentage of identityis calculated over the full length of the reference amino acid sequenceand that gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed.

In a specific embodiment, the identity between a reference (query)sequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, is determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDBamino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1,Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, WindowSize=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, WindowSize=500 or the length of the subject amino acid sequence, whichever isshorter. According to this embodiment, if the subject sequence isshorter than the query sequence due to N- or C-terminal deletions, notbecause of internal deletions, a manual correction is made to theresults to take into consideration the fact that the FASTDB program doesnot account for N- and C-terminal truncations of the subject sequencewhen calculating global percent identity. For subject sequencestruncated at the N- and C-termini, relative to the query sequence, thepercent identity is corrected by calculating the number of residues ofthe query sequence that are N- and C-terminal of the subject sequence,which are not matched/aligned with a corresponding subject residue, as apercent of the total bases of the query sequence. A determination ofwhether a residue is matched/aligned is determined by results of theFASTDB sequence alignment. This percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thisfinal percent identity score is what is used for the purposes of thisembodiment. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence. For example, a 90 aminoacid residue subject sequence is aligned with a 100 residue querysequence to determine percent identity. The deletion occurs at theN-terminus of the subject sequence and therefore, the FASTDB alignmentdoes not show a matching/alignment of the first 10 residues at theN-terminus. The 10 unpaired residues represent 10% of the sequence(number of residues at the N- and C-termini not matched/total number ofresidues in the query sequence) so 10% is subtracted from the percentidentity score calculated by the FASTDB program. If the remaining 90residues were perfectly matched the final percent identity would be 90%.In another example, a 90 residue subject sequence is compared with a 100residue query sequence. This time the deletions are internal deletionsso there are no residues at the N- or C-termini of the subject sequencewhich are not matched/aligned with the query. In this case the percentidentity calculated by FASTDB is not manually corrected. Once again,only residue positions outside the N- and C-terminal ends of the subjectsequence, as displayed in the FASTDB alignment, which are notmatched/aligned with the query sequence are manually corrected for. Noother manual corrections are made for the purposes of this embodiment.

The polypeptides of the present invention include the polypeptide of SEQID NO:2 (in particular the mature polypeptide) as well as polypeptideswhich have at least 70% similarity (preferably at least 70% identity) tothe polypeptide of SEQ ID NO:2 and more preferably at least 90%similarity (more preferably at least 90% identity) to the polypeptide ofSEQ ID NO:2 and still more preferably at least 95% similarity (stillmore preferably at least 95% identity) to the polypeptide of SEQ ID NO:2and also include portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

The polypeptides of the present invention also include the polypeptideof SEQ ID NO:20 (in particular the extracellular domain of thepolypeptide) as well as polypeptides, which have at least 70% similarity(preferably at least 70% identity) to the polypeptide of SEQ ID NO:20and more preferably at least 90% similarity (more preferably at least90% identity) to the polypeptide of SEQ ID NO:20 and still morepreferably at least 95% similarity (still more preferably at least 95%identity) to the polypeptide of SEQ ID NO:20 and also include portionsof such polypeptides with such portion of the polypeptide generallycontaining at least 30 amino acids and more preferably at least 50 aminoacids.

Further polypeptides of the present invention include polypeptides haveat least 70% similarity, at least 90% similarity, more preferably atleast 95% similarity, and still more preferably at least 96%, 97%, 98%or 99% similarity to those polypeptides described herein. Thepolypeptides of the invention also comprise those which are at least 70%identical, at least 80% identical, more preferably at least 90% or 95%identical, still more preferably at least 96%, 97%, 98% or 99% identicalto the polypeptides disclosed herein. In specific embodiments, suchpolypeptides comprise at least 30 amino acids and more preferably atleast 50 amino acids.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.By “% similarity” for two polypeptides is intended a similarity scoreproduced by comparing the amino acid sequences of the two polypeptidesusing the Bestfit program (Wisconsin Sequence Analysis Package, Version8 for Unix, Genetics Computer Group, University Research Park, 575Science Drive, Madison, Wis. 53711) and the default settings fordetermining similarity. Bestfit uses the local homology algorithm ofSmith and Waterman (Advances in Applied Mathematics 2:482-489, 1981) tofind the best segment of similarity between two sequences.

The TNF-gamma-alpha and TNF-gamma-beta polypeptides of the invention maybe in monomers or multimers (i.e., dimers, trimers, tetramers and highermultimers). In specific embodiments, the polypeptides of the inventionare monomers, dimers, trimers or tetramers. In additional embodiments,the multimers of the invention are at least dimers, at least trimers, orat least tetramers.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term homomer, refers to a multimer containing onlyTNF-gamma-alpha and/or TNF-gamma-beta polypeptides of the invention(including TNF-gamma-alpha and/or TNF-gamma-beta fragments, variants,and fusion proteins, as described herein). These homomers may containTNF-gamma-alpha and TNF-gamma-beta polypeptides having identical ordifferent amino acid sequences. In a specific embodiment, a homomer ofthe invention is a multimer containing only TNF-gamma-alpha and/orTNF-gamma-beta polypeptides having an identical amino acid sequence. Inanother specific embodiment, a homomer of the invention is a multimercontaining TNF-gamma-alpha and TNF-gamma-beta polypeptides havingdifferent amino acid sequences. In specific embodiments, the multimer ofthe invention is a homodimer (e.g., containing TNF-gamma-alphapolypeptides having identical or different amino acid sequences) or ahomotrimer (e.g., containing TNF-gamma-alpha polypeptides havingidentical or different amino acid sequences). In additional embodiments,the homomeric multimer of the invention is at least a homodimer, atleast a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containingheterologous polypeptides (i.e., polypeptides of a different protein) inaddition to the TNF-gamma-alpha and TNF-gamma-beta polypeptides of theinvention. In a specific embodiment, the multimer of the invention is aheterodimer, a heterotrimer, or a heterotetramer. In additionalembodiments, the homomeric multimer of the invention is at least ahomodimer, at least a homotrimer, or at least a homotetramer.

Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations. Thus, in oneembodiment, multimers of the invention, such as, for example, homodimersor homotrimers, are formed when polypeptides of the invention contactone another in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentinteractions with and/or between the TNF-gamma-alpha and TNF-gamma-betapolypeptides of the invention. Such covalent interactions may involveone or more amino acid residues corresponding to those recited in SEQ IDNO:2 or SEQ ID NO:20, or corresponding to one or more amino acidresidues encoded by the clones HUVEO91 and HEMCZ56, respectively.Alternatively, such covalent interactions may involve one or more aminoacid residues contained in the heterologous polypeptide sequence in aTNF-gamma-alpha and TNF-gamma-beta fusion protein, such as for example,heterologous sequence contained in a TNF-gamma-alpha-Fc fusion protein(as described herein), and heterologous sequence contained in a fusionwith heterologous polypeptide sequence from another TNF familyligand/receptor member, such as, for example, osteoprotegerin, that iscapable of forming covalently associated multimers.

The invention also encompasses fusion proteins in which the full lengthTNF-gamma polypeptide or fragment, variant, derivative, or analogthereof is fused to an unrelated protein. Fusion proteins of theinvention may be constructed as direct fusion of TNF-gamma polypeptide(or fragment, variant, derivative, or analog) and a heterologoussequence, or may be constructed with a spacer or adapter region havingone or more amino acids inserted between the two portions of theprotein. Optionally, the spacer region may encode a protease cleavagesite. The precise site of the fusion is not critical and may beroutinely varied by one skilled in the art in order to maximize bindingcharacteristics and/or biological activity of the homologous and/orheterologous sequence(s). The fusion proteins of the invention can beroutinely designed on the basis of the TNF-gamma nucleotide andpolypeptide sequences disclosed herein. For example, as one of skill inthe art will appreciate, TNF-gamma-alpha and/or TNF-gamma-betapolypeptides and fragments (including epitope-bearing fragments) thereofdescribed herein can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric (fusion) polypeptides.These fusion proteins facilitate purification and show an increasedhalf-life in vivo. This has been shown, e.g., for chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions of the heavy or light chains ofmammalian immunoglobulins (EP A 394,827; Traunecker, et aL, Nature331:84-86 (1988)). Fusion proteins that have a disulfide-linked dimericstructure due to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric TNF-gamma protein orprotein fragment alone (Fountoulakis, et al., J. Biochem. 270:3958-3964(1995)). As an example, one such TNF-gamma-Fc fusion has been producedherein as described above. In other embodiments, the full lengthTNF-gamma polypeptide or fragment, variant, derivative, or analogthereof is fused to one or more other heterologous polypeptide sequencesthat are capable of forming multimeric formations, such as, for example,the dimerization domain of osteoprotegrin (see, e.g,. EP 0 721 983, U.S.Pat. No. 5,478,925, and International Publication No. WO 98/49305, eachof which is herein incorporated by reference in its entirety).Additional examples of TNF-gamma fusion proteins that are encompassed bythe invention include, but are not limited to, fusion of the TNF-gammapolypeptide sequence to any amino acid sequence that allows the fusionprotein to be displayed on the cell surface; or fusions to an enzyme,fluorescent protein, or luminescent protein which provides a markerfunction.

Modifications of chimeric OPG polypeptides are encompassed by theinvention and include post-translational modifications (e.g., N-linkedor O-linked carbohydrate chains, processing of N-terminal or C-terminalends), attachment of chemical moieties to the amino acid backbone,chemical modifications of N-linked or O-linked carbohydrate chains, andaddition of an N-terminal methionine residue as a result of procaryotichost cell expression. The polypeptides may also be modified with adetectable label, such as an enzymatic, fluorescent, isotopic oraffinity label to allow for detection and isolation of the protein.

Also provided by the invention are chemically modified derivatives ofOPG which may provide additional advantages such as increasedsolubility, stability and circulating time of the polypeptide, ordecreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemicalmoieties for derivitization may be selected from water soluble polymerssuch as polyethylene glycol, ethylene glycol/propylene glycolcopolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and thelike. The polypeptides may be modified at random positions within themolecule, or at predetermined positions within the molecule and mayinclude one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (or peptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

The polypeptides of the present invention have uses which include, butare not limited to, molecular weight marker on SDS-PAGE gels or onmolecular sieve gel filtration columns using methods well known to thoseof skill in the art.

Functional Activities

The functional activity of TNF-gamma polypeptides, and fragments,variants derivatives, and analogs thereof can be assayed by variousmethods.

For example, in one embodiment where one is assaying for the ability tobind or compete with full-length TNF-gamma polypeptide for binding toanti-TNF-gamma antibody, various immunoassays known in the art can beused, including but not limited to, competitive and non-competitiveassay systems using techniques such as radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays, immunoradiometricassays, gel diffusion precipitation reactions, immunodiffusion assays,in situ immunoassays (using colloidal gold, enzyme or radioisotopelabels, for example), western blots, precipitation reactions,agglutination assays (e.g., gel agglutination assays, hemagglutinationassays), complement fixation assays, immunofluorescence assays, proteinA assays, and immunoelectrophoresis assays, etc. In one embodiment,antibody binding is detected by detecting a label on the primaryantibody. In another embodiment, the primary antibody is detected bydetecting binding of a secondary antibody or reagent to the primaryantibody. In a further embodiment, the secondary antibody is labelled.Many means are known in the art for detecting binding in an immunoassayand are within the scope of the present invention.

In another embodiment, where a TNF-ligand is identified, binding can beassayed, e.g., by means well-known in the art. In another embodiment,physiological correlates of TNF-gamma binding to its substrates (signaltransduction) can be assayed.

In addition, assays described herein (see Examples 5, 6, and 9-15, andotherwise known in the art may routinely be applied to measure theability of TNF-gamma polypeptides and fragments, variants derivativesand analogs thereof to elicit TNF-gamma related biological activity(e.g., to inhibit, or alternatively promote, cell proliferation, tumorformation, angiogenesis, NF-κB activation and cell adhesion in vitro orin vivo).

Other methods will be known to the skilled artisan and are within thescope of the invention.

Antibodies

The present invention further relates to antibodies and T-cell antigenreceptors (TCR) which specifically bind the polypeptides of the presentinvention. The antibodies of the present invention include IgG(including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2),IgD, IgE, or IgM, and IgY. As used herein, the term “antibody” (Ab) ismeant to include whole antibodies, including single-chain wholeantibodies, and antigen-binding fragments thereof. Most preferably theantibodies are human antigen binding antibody fragments of the presentinvention include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd,single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs(sdFv) and fragments comprising either a V_(L) or V_(H) domain. Theantibodies may be from any animal origin including birds and mammals.Preferably, the antibodies are human, murine, rabbit, goat, guinea pig,camel, horse, or chicken.

Antigen-binding antibody fragments, including single-chain antibodies,may comprise the variable region(s) alone or in combination with theentire or partial of the following: hinge region, CH1, CH2, and CH3domains. Also included in the invention are any combinations of variableregion(s) and hinge region, CH1, CH2, and CH3 domains. The presentinvention further includes chimeric, humanized, and human monoclonal andpolyclonal antibodies which specifically bind the polypeptides of thepresent invention. The present invention further includes antibodieswhich are anti-idiotypic to the antibodies of the present invention.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for heterologous compositions, such as aheterologous polypeptide or solid support material. See, e.g., WO93/17715; WO 92/08802; WO 91/00360; WO 92105793; Tutt, A. et al. (1991)J. Immunol. 147:60-69; U.S. Pat. Nos. 5,573,920, 4,474,893, 5,601,819,4,714,681, 4,925,648; Kostelny, S. A. et al. (1992) J. Immunol.148:1547-1553.

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which are recognized or specifically bound by the antibody.The epitope(s) or polypeptide portion(s) may be specified as describedherein, e.g., by N-terminal and C-terminal positions, by size incontiguous amino acid residues, or listed in the Tables and Figures.Antibodies which specifically bind any epitope or polypeptide of thepresent invention may also be excluded. Therefore, the present inventionincludes antibodies that specifically bind polypeptides of the presentinvention, and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homolog of the polypeptides of the presentinvention are included. Antibodies that do not bind polypeptides withless than 95%, less than 90%, less than 85%, less than 80%, less than75%, less than 70%, less than 65%, less than 60%, less than 55%, andless than 50% identity (as calculated using methods known in the art anddescribed herein) to a polypeptide of the present invention are alsoincluded in the present invention. Further included in the presentinvention are antibodies which only bind polypeptides encoded bypolynucleotides which hybridize to a polynucleotide of the presentinvention under stringent hybridization conditions (as describedherein). Antibodies of the present invention may also be described orspecified in terms of their binding affinity. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M, 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M,5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹M, 10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M,5×10⁻¹⁴M, 10⁻¹⁴M, 5×10⁻¹⁵M, and 10⁻¹⁵M.

Antibodies of the present invention have uses that include, but are notlimited to, methods known in the art to purify, detect, and target thepolypeptides of the present invention including both in vitro and invivo diagnostic and therapeutic methods. For example, the antibodieshave use in immunoassays for qualitatively and quantitatively measuringlevels of the polypeptides of the present invention in biologicalsamples. See, e.g., Harlow et al., ANTIBODIES: A LABORATORY MANUAL,(Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated byreference in the entirety).

The antibodies of the present invention may be used either alone or incombination with other compositions. The antibodies may further berecombinantly fused to a heterologous polypeptide at the N- orC-terminus or chemically conjugated (including covalently andnon-covalently conjugations) to polypeptides or other compositions. Forexample, antibodies of the present invention may be recombinantly fusedor conjugated to molecules useful as labels in detection assays andeffector molecules such as heterologous polypeptides, drugs, or toxins.See, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 0 396 387.

The antibodies of the present invention may be prepared by any suitablemethod known in the art. For example, a polypeptide of the presentinvention or an antigenic fragment thereof can be administered to ananimal in order to induce the production of sera containing polyclonalantibodies. Monoclonal antibodies can be prepared using a wide oftechniques known in the art including the use of hybridoma andrecombinant technology. See, e.g., Harlow et al., ANTIBODIES: ALABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);Hammerling, et al., in: MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS563-681 (Elsevier, N.Y., 1981) (said references incorporated byreference in their entireties).

Fab and F(ab′)2 fragments may be produced by proteolytic cleavage, usingenzymes such as papain (to produce Fab fragments) or pepsin (to produceF(ab′)2 fragments).

Alternatively, antibodies of the present invention can be producedthrough the application of recombinant DNA technology or throughsynthetic chemistry using methods known in the art. For example, theantibodies of the present invention can be prepared using various phagedisplay methods known in the art. In phage display methods, functionalantibody domains are displayed on the surface of a phage particle whichcarries polynucleotide sequences encoding them. Phage with a desiredbinding property are selected from a repertoire or combinatorialantibody library (e.g. human or murine) by selecting directly withantigen, typically antigen bound or captured to a solid surface or bead.Phage used in these methods are typically filamentous phage including fdand M13 with Fab, Fv or disulfide stabilized Fv antibody domainsrecombinantly fused to either the phage gene III or gene VIII protein.Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in BrinkmanU. et al. (1995) J. Immunol. Methods 182:41-50; Ames, R. S. et al.(1995) J. Immunol. Methods 184:177-186; Kettleborough, C. A. et al.(1994) Eur. J. Immunol. 24:952-958; Persic, L. et al. (1997) Gene 1879-18; Burton, D. R. et al. (1994) Advances in Immunology 57:191-280;PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426,5,223,409, 5,463,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047,5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743(said references incorporated by reference in their entireties).

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired hostincluding mammalian cells, insect cells, plant cells, yeast, andbacteria. For example, techniques to recombinantly produce Fab, Fab′ andF(ab′)2 fragments can also be employed using methods known in the artsuch as those disclosed in WO 92/22324; Mullinax, R. L. et al. (1992)BioTechniques, 12(6):864-869; and Sawai, H. et al. (1995) AJRI 34:26-34;and Better, M. et al. (1988) Science 240:1041-1043 (said referencesincorporated by reference in their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al. (1991) Methods in Enzymology 203:46-88; Shu, L.et al. (1993) PNAS 90:7995-7999; and Skerra, A. et al. (1988) Science240:1038-1040. For some uses, including in vivo use of antibodies inhumans and in vitro detection assays, it may be preferable to usechimeric, humanized, or human antibodies. Methods for producing chimericantibodies are known in the art. See e.g., Morrison, Science 229:1202(1985); Oi et al., BioTechniques 4:214 (1986); Gillies, S. D. et al.(1989) J. Immunol. Methods 125:191-202; and U.S. Pat. No. 5,807,715.Antibodies can be humanized using a variety of techniques includingCDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. Nos. 5,530,101; and5,585,089), veneering or resurfacing (EP 0 592 106; EP 0 519 596; PadlanE. A., (1991) Molecular Immunology 28(4/5):489-498; Studnicka G. M. etal. (1994) Protein Engineering 7(6):805-814; Roguska M. A. et al. (1994)PNAS 91:969-973), and chain shuffling (U.S. Pat. No. 5,565,332). Humanantibodies can be made by a variety of methods known in the artincluding phage display methods described above. See also, U.S. Pat.Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; and WO 98/46645(said references incorporated by reference in their entireties).

Further included in the present invention are antibodies recombinantlyfused or chemically conjugated (including both covalently andnon-covalently conjugations) to a polypeptide of the present invention.The antibodies may be specific for antigens other than polypeptides ofthe present invention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal. supra and WO 93/21232; EP 0 439 095; Naramura, M. et al. (1994)Immunol. Lett. 39:91-99; U.S. Pat. No. 5,474,981; Gillies, S. O. et al.(1992) PNAS 89:1428-1432; Fell, H. P. et al. (1991) J. Immunol.146:2446-2452 (said references incorporated by reference in theirentireties).

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the hinge region, CH1 domain, CH2domain, and CH3 domain or any combination of whole domains or portionsthereof. The polypeptides of the present invention may be fused orconjugated to the above antibody portions to increase the in vivo halflife of the polypeptides or for use in immunoassays using methods knownin the art. The polypeptides may also be fused or conjugated to theabove antibody portions to form multimers. For example, Fc portionsfused to the polypeptides of the present invention can form dimersthrough disulfide bonding between the Fc portions. Higher multimericforms can he made by fusing the polypeptides to portions of IgA and IgM.Methods for fusing or conjugating the polypeptides of the presentinvention to antibody portions are known in the art. See e.g., U.S. Pat.Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,112,946;EP 0 307 434, EP 0 367 166; WO 96/04388, WO 91/06570; Ashkenazi, A. etal. (1991) PNAS 88:10535-10539; Zheng, X. X. et al. (1995) J. Immunol.154:5590-5600; and Vil, H. et al. (1992) PNAS 89:11337-11341 (saidreferences incorporated by reference in their entireties).

The invention further relates to antibodies which act as agonists orantagonists of the polypeptides of the present invention. For example,the present invention includes antibodies which disrupt thereceptor/ligand interactions with the polypeptides of the inventioneither partially or fully. Included are both receptor-specificantibodies and ligand-specific antibodies. Included arereceptor-specific antibodies which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. Also include are receptor-specific antibodies which both preventligand binding and receptor activation. Likewise, included areneutralizing antibodies which bind the ligand and prevent binding of theligand to the receptor, as well as antibodies which bind the ligand,thereby preventing receptor activation, but do not prevent the ligandfrom binding the receptor. Further included are antibodies whichactivate the receptor. These antibodies may act as agonists for toeither all or less than all of the biological activities affected byligand-mediated receptor activation. The antibodies may be specified asagonists or antagonists for biological activities comprising specificactivities disclosed herein. The above antibody agonists can be madeusing methods known in the art. See e.g., WO 96/40281; U.S. Pat. No.5,811,097; Deng, B. et al. (1998) Blood 92(6):1981-1988; Chen, Z. et al.(1998) Cancer Res. 58(16):3668-3678; Harrop, J. A. et al. (1998) J.Immunol. 161(4):1786-1794; Zhu, Z. et al. (1998) Cancer Res.58(15):3209-3214; Yoon, D. Y. et al. (1998) J. Immunol.160(7):3170-3179; Prat, M. et al. (1998) J. Cell. Sci. 111(Pt2):237-247;Pitard, V. et al. (1997) J. Immunol. Methods 205(2):177-190; Liautard,J. et al. (1997) Cytokinde 9(4):233-241; Carlson, N. G. et al. (1997) J.Biol. Chem. 272(17):11295-11301; Taryman, R. E. et al. (1995) Neuron14(4):755-762; Muller, Y. A. et al. (1998) Structure 6(9):1153-1167;Bartunek, P. et al. (1996) Cytokine 8(1): 14-20 (said referencesincorporated by reference in their entireties).

Transgenics

The polypeptides of the invention can also be expressed in transgenicanimals. Animals of any species, including, but not limited to, mice,rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep,cows and non-human primates, e.g., baboons, monkeys, and chimpanzees maybe used to generate transgenic animals. In a specific embodiment,techniques described herein or otherwise known in the art, are used toexpress polypeptides of the invention in humans, as part of a genetherapy protocol.

Any technique known in the art may be used to introduce the transgene(i.e., polynucleotides of the invention) into animals to produce thefounder lines of transgenic animals. Such techniques include, but arenot limited to, pronuclear microinjection ((each of the followingreferences is hereby incorporated by reference) Paterson et al., Appl.Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology(N.Y.) 11:1263-1270 (1993); Wright et al., Biotechnology (N.Y.)9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989));retrovirus mediated gene transfer into germ lines ((the followingreference is hereby incorporated by reference) VanderPutten et al.,Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts orembryos; gene targeting in embryonic stem cells ((each of the followingreferences is hereby incorporated by reference) Thompson et al., Cell56:313-321 (1989)); electroporation of cells or embryos (Lo, 1983, MolCell. Biol. 3:1803-1814 (1983)); introduction of the polynucleotides ofthe invention using a gene gun ((the following reference is herebyincorporated by reference) see, e.g., Ulmer et al., Science 259:1745(1993); introducing nucleic acid constructs into embryonic pleuripotentstem cells and transferring the stem cells back into the blastocyst; andsperm-mediated gene transfer ((the following reference is herebyincorporated by reference) Lavitrano et al., Cell 57:717-723 (1989);etc. For a review of such techniques, see Gordon, “Transgenic Animals,”Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by referenceherein in its entirety.

Any technique known in the art may be used to produce transgenic clonescontaining polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence ((each of the followingreferences is hereby incorporated by reference) Campell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric. The transgene may be integrated as a single transgene or asmultiple copies such as in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al. ((the following reference is herebyincorporated by reference) Lasko et al., Proc. Natl. Acad. Sci. USA89:6232-6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide transgene be integrated into thechromosomal site of the endogenous gene, gene targeting is preferred.Briefly, when such a technique is to be utilized, vectors containingsome nucleotide sequences homologous to the endogenous gene are designedfor the purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of thenucleotide sequence of the endogenous gene. The transgene may also beselectively introduced into a particular cell type, thus inactivatingthe endogenous gene in only that cell type, by following, for example,the teaching of Gu et al. ((the following reference is herebyincorporated by reference) Gu et al., Science 265:103-106 (1994)). Theregulatory sequences required for such a cell-type specific inactivationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art.

Once transgenic animals have been generated the expression of therecombinant gene may be assayed utilizing standard techniques. Initialscreening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines lo produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

Transgenic and “knock-out” animals of the invention have uses whichinclude, but are not limited to, animal model systems useful inelaborating the biological function of TNF-gamma-alpha and/orTNF-gamma-beta polypeptides, studying conditions and/or disordersassociated with aberrant TNF-gamma-alpha and/or TNF-gamma-betaexpression, and in screening for compounds effective in amelioratingsuch conditions and/or disorders.

Endogenous gene expression can also be reduced by inactivating or“knocking out” the gene and/or its promoter using targeted homologousrecombination. ((each of the following references is hereby incorporatedby reference) E.g., see Smithies et al., Nature 317:230-234 (1985);Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell5:313-321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene ((each of the followingreferences is hereby incorporated by reference) e.g. see Thomas &Capecchi 1987 and Thompson 1989, supra). However this approach can beroutinely adapted for use in humans provided the recombinant DNAconstructs are directly administered or targeted to the required site invivo using appropriate viral vectors that will be apparent to those ofskill in the art.

In further embodiments of the invention, cells that are geneticallyengineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.,lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cellsare genetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe polypeptides of the invention. The engineered cells which expressand preferably secrete the polypeptides of the invention can beintroduced into the patient systemically, e.g., in the circulation, orintraperitoneally. Alternatively, the cells can be incorporated into amatrix and implanted in the body, e.g., genetically engineeredfibroblasts can be implanted as part of a skin graft; geneticallyengineered endothelial cells can be implanted as part of a lymphatic orvascular graft. ((each of the following references is herebyincorporated by reference) See, for example, Anderson et al. U.S. Pat.No. 5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959 each ofwhich is incorporated by reference herein in its entirety).

When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

Immune and Circulatory Systems-Related Disorders

Diagnosis

The present inventors have discovered that TNF-gamma is expressed inhuman umbilical vein endothelial cells, induced endothelial cells,macrophages, and substantia nigra tissue. For a number of immune andcirculatory systems-related disorders, substantially altered (increasedor decreased) levels of TNF-gamma-alpha and/or TNF-gamma-beta geneexpression can be detected in immune and circulatory systems tissue orother cells or bodily fluids (e.g., sera, plasma, urine, synovial fluidor spinal fluid) taken from an individual having such a disorder,relative to a “standard” TNF-gamma-alpha and/or TNF-gamma-beta geneexpression level, that is, the TNF-gamma-alpha and/or TNF-gamma-betaexpression level in immune and circulatory systems tissues or bodilyfluids from an individual not having the immune and circulatory systemsdisorder. Thus, the invention provides a diagnostic method useful duringdiagnosis of a immune and circulatory systems disorder, which involvesmeasuring the expression level of the gene encoding the TNF-gamma-alphaand/or TNF-gamma-beta protein in immune and circulatory systems tissueor other cells or body fluid from an individual and comparing themeasured gene expression level with a standard TNF-gamma-alpha and/orTNF-gamma-beta gene expression level, whereby an increase or decrease inthe gene expression level compared to the standard is indicative of animmune and circulatory systems disorder.

In particular, it is believed that certain tissues in mammals withcancer of the immune and circulatory systems express significantlyreduced levels of the TNF-gamma-alpha and/or TNF-gamma-beta protein andmRNA encoding the TNF-gamma-alpha and/or TNF-gamma-beta protein whencompared to a corresponding “standard” level. Further, it is believedthat enhanced levels of the TNF-gamma-alpha and/or TNF-gamma-betaprotein can be detected in certain body fluids (e.g., sera, plasma,urine, and spinal fluid) from mammals with such a cancer when comparedto sera from mammals of the same species not having the cancer.

Thus, the invention provides a diagnostic method useful during diagnosisof a immune and circulatory systems disorder, including cancers of thesesystems, which involves measuring the expression level of the geneencoding the TNF-gamma-alpha and/or TNF-gamma-beta protein in immune andcirculatory systems tissue or other cells or body fluid from anindividual and comparing the measured gene expression level with astandard TNF-gamma-alpha and/or TNF-gamma-beta gene expression level,whereby an increase or decrease in the gene expression level compared tothe standard is indicative of an immune and circulatory systemsdisorder.

Where a diagnosis of a disorder in the immune and circulatory systems,including diagnosis of a tumor, has already been made according toconventional methods, the present invention is useful as a prognosticindicator, whereby patients exhibiting depressed TNF-gamma-alpha and/orTNF-gamma-beta gene expression will experience a worse clinical outcomerelative to patients expressing the gene at a level nearer the standardlevel.

By “assaying the expression level of the gene encoding theTNF-gamma-alpha and/or TNF-gamma-beta protein” is intended qualitativelyor quantitatively measuring or estimating the level of theTNF-gamma-alpha and/or TNF-gamma-beta protein or the level of the mRNAencoding the TNF-gamma-alpha and/or TNF-gamma-beta protein in a firstbiological sample either directly (e.g., by determining or estimatingabsolute protein level or mRNA level) or relatively (e.g., by comparingto the TNF-gamma-alpha and/or TNF-gamma-beta protein level or mRNA levelin a second biological sample). Preferably, the TNF-gamma-alpha and/orTNF-gamma-beta protein level or mRNA level in the first biologicalsample is measured or estimated and compared to a standardTNF-gamma-alpha and/or TNF-gamma-beta protein level or mRNA level, thestandard being taken from a second biological sample obtained from anindividual not having the disorder or being determined by averaginglevels from a population of individuals not having a disorder of theimmune and circulatory systems. As will be appreciated in the art, oncea standard TNF-gamma-alpha and/or TNF-gamma-beta protein level or mRNAlevel is known, it can be used repeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, body fluid, cell line, tissue culture, or other sourcewhich contains TNF-gamma-alpha and/or TNF-gamma-beta protein or mRNA. Asindicated, biological samples include body fluids (such as sera, plasma,urine, synovial fluid and spinal fluid) which contain freeTNF-gamma-alpha and/or TNF-gamma-beta protein, immune and circulatorysystems tissue, and other tissue sources found to express complete ormature TNF-gamma-alpha and/or TNF-gamma-beta or a TNF-gamma-alpha and/orTNF-gamma-beta receptor. Methods for obtaining tissue biopsies and bodyfluids from mammals are well known in the art. Where the biologicalsample is to include mRNA, a tissue biopsy is the preferred source.

Total cellular RNA can be isolated from a biological sample using anysuitable technique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described byChomczynski and Sacchi (Anal. Biochem. 162:156-159 (1987)). Levels ofmRNA encoding the TNF-gamma-alpha and/or TNF-gamma-beta protein are thenassayed using any appropriate method. These include Northern blotanalysis, S1 nuclease mapping, the polymerase chain reaction (PCR),reverse transcription in combination with the polymerase chain reaction(RT-PCR), and reverse transcription in combination with the ligase chainreaction (RT-LCR).

Assaying TNF-gamma-alpha and/or TNF-gamma-beta protein levels in abiological sample can occur using antibody-based techniques. Forexample, TNF-gamma-alpha and/or TNF-gamma-beta protein expression intissues can be studied with classical immunohistological methods(Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M.,et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-basedmethods useful for detecting TNF-gamma-alpha and/or TNF-gamma-betaprotein gene expression include immunoassays, such as the enzyme linked,immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitableantibody assay labels are known in the art and include enzyme labels,such as, glucose oxidase, and radioisotopes, such as iodine (¹²⁵I,¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹²In), andtechnetium (^(99m)Tc), and fluorescent labels, such as fluorescein andrhodamine, and biotin.

In addition to assaying TNF-gamma-alpha and/or TNF-gamma-beta proteinlevels in a biological sample obtained from an individual,TNF-gamma-alpha and/or TNF-gamma-beta protein can also be detected invivo by imaging. Antibody labels or markers for in viva imaging ofTNF-gamma-alpha and/or TNF-gamma-beta protein include those detectableby X-radiography, NMR or ESR. For X-radiography, suitable labels includeradioisotopes such as barium or cesium, which emit detectable radiationbut are not overtly harmful to the subject. Suitable markers for NMR andESR include those with a detectable characteristic spin, such asdeuterium, which may be incorporated into the antibody by labeling ofnutrients for the relevant hybridoma.

A TNF-gamma-alpha and/or TNF-gamma-beta protein-specific antibody orantibody fragment which has been labeled with an appropriate detectableimaging moiety, such as a radioisotope (for example, ¹³¹I, ¹¹²In,^(99n)Tc), a radio-opaque substance, or a material detectable by nuclearmagnetic resonance, is introduced (for example, parenterally,subcutaneously or intraperitoneally) into the mammal to be examined forimmune system disorder. It will be understood in the art that the sizeof the subject and the imaging system used will determine the quantityof imaging moiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of ^(99m)Tc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain TNF-gamma-alpha and/orTNF-gamma-beta protein. In vivo tumor imaging is described by Burchieland coworkers (Chapter 13 in Tumor Imaging: The Radiochemical Detectionof Cancer, Burchiel, S. W. and Rhodes, B. A., eds., Masson PublishingInc. (1982)).

Treatment

As noted above, TNF-gamma-alpha and/or TNF-gamma-beta polynucleotidesand polypeptides are useful for diagnosis of conditions involvingabnormally high or low expression of TNF-gamma-alpha and/orTNF-gamma-beta activities. Given the cells and tissues whereTNF-gamma-alpha and/or TNF-gamma-beta is expressed as well as theactivities modulated by TNF-gamma-alpha and/or TNF-gamma-beta, it isreadily apparent that a substantially altered (increased or decreased)level of expression of TNF-gamma-alpha and/or TNF-gamma-beta in anindividual compared to the standard or “normal” level producespathological conditions related to the bodily system(s) in whichTNF-gamma-alpha and/or TNF-gamma-beta is expressed and/or is active.

It is well-known in the art that, in addition to a specific cellularfunction, cellular receptor molecules may also often be exploited by avirus as a means of initiating entry into a potential host cell. Forexample, it was recently discovered by Wu and colleagues (J. Exp. Med.185:1681-1691 (1997)) that the cellular chemokine receptor CCR5functions not only as a cellular chemokine receptor, but also as areceptor for macrophage-tropic human immunodeficiency virus (HIV)-1. Inaddition, RANTES, MIP-1a, and MIP-1b, which are agonists for thecellular chemokine receptor CCR5, inhibit entry of various strains ofHIV-1 into susceptible cell lines (Cocchi, F., et al., Science270:1811-1815 (1995)). Thus, the invention also provides a method oftreating an individual exposed to, or infected with, a virus through theprophylactic or therapeutic administration of TNF-gamma-alpha and/orTNF-gamma-beta, or an agonist or antagonist thereof, to block or disruptthe interaction of a viral particle with the TNF-gamma-alpha and/orTNF-gamma-beta receptor and, as a result, block the initiation orcontinuation of viral infectivity.

The TNF-gamma-alpha and/or TNF-gamma-beta of the present invention bindsto the TNF-gamma-alpha and/or TNF-gamma-beta receptor and, as such, islikely to block immuno- and endothelial cell-tropic viral infections.Expression patterns of the cDNA clone encoding the present inventionsuggests that this molecule is expressed primarily in endothelial cellsand select hematopoietic tissues. When considered together, theseobservations suggest that agonists and antagonists, including areceptor, of TNF-gamma-alpha and/or TNF-gamma-beta may be useful as amethod of blocking or otherwise regulating the infectivity ofimmunotropic viral infections. A non-limiting list of viruses whichinfect humans and can infect cells of the hematopoietic system includessuch retroviruses as HIV-1, HIV-2, human T-cell lymphotropic virus(HTLV)-I, and HTLV-II, as well as other DNA and RNA viruses such asherpes simplex virus (HSV)-1, HSV-2, HSV-6, cytomegalovirus (CMV),Epstein-Barr virus (EBV), herpes samirii, adenoviruses, rhinoviruses,influenza viruses, reoviruses, and the like.

The ability of TNF-gamma-alpha and/or TNF-gamma-beta of the presentinvention, or agonists or antagonists thereof, to prophylactically ortherapeutically block viral infection may be easily tested by theskilled artisan. For example, Simmons and coworkers (Science 276:276-279(1997)) and Arenzana-Seisdedos and colleagues (Nature 383;400 (1996))each outline a method of observing suppression of HIV-1, infection by anantagonist of the CCR5 chemokine receptor and of the CC chemokineRANTES, respectively, in cultured peripheral blood mononuclear cells.Cells are cultured and infected with a virus, HIV-1 in both cases notedabove. An agonist or antagonist of the CC chemokine or its receptor isthen immediately added to the culture medium. Evidence of the ability ofthe agonist or antagonist of the chemokine or cellular receptor isdetermined by evaluating the relative success of viral infection at 3,6, and 9 days postinfection.

Administration of a pharmaceutical composition comprising an amount ofan isolated TNF-gamma-alpha and/or TNF-gamma-beta, or an agonist orantagonist thereof, of the invention to an individual either infectedwith a virus or at risk for infection with a virus is performed asdescribed below.

The present invention is also useful for diagnosis or treatment ofvarious immune and circulatory system-related disorders in mammals,preferably humans. Such disorders include tumors (an incomplete list ofhuman tumors includes breast cancer, colon cancer, cardiac tumors,pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer,intestinal cancer, testicular cancer, stomach cancer, neuroblastoma,myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma,adenoma, and the like) and tumor metastasis, infections by bacteria,viruses, and other parasites, immunodeficiencies, inflammatory diseases,lymphadenopathy, autoimmune diseases, graft versus host disease, and anydisregulation of immune and circulatory systems cell function including,but not limited to, autoimmunity, arthritis, leukemias, lymphomas,immunosuppression, immunity, humoral immunity, inflammatory boweldisease, myelo suppression, and the like.

Since TNF-gamma has been shown to induce activation of cellular NF-kBand c-jun N-terminal kinase (JNK), it is also useful in therapeuticallyregulating such cellular and immune systemic disorders as tumors andtumor metastases, infections by bacteria, viruses, and other parasites,immunodeficiencies, inflammatory diseases, lymphadenopathy, autoimmunediseases, graft versus host disease, autoimmunity, arthritis, leukemias,lymphomas, immunosuppression, inflammatory bowel disease,myelosuppression, and related sequelae.

Since TNF-gamma-alpha and TNF-gamma-beta belong to the TNF superfamily,they also also modulate angiogenesis. In addition, since TNF-gamma-alphaand/or TNF-gamma-beta inhibit immune cell functions, it will have a widerange of anti-inflammatory activities. TNF-gamma-alpha and/orTNF-gamma-beta may be employed as an anti-neovascularizing agent totreat solid tumors by stimulating the invasion and activation of hostdefense cells, e.g., cytotoxic T-cells and macrophages and by inhibitingthe angiogenesis of tumors. Those of skill in the art will recognizeother non-cancer indications where blood vessel proliferation is notwanted. They may also be employed to enhance host defenses againstresistant chronic and acute infections, for example, myobacterialinfections via the attraction and activation of microbicidal leukocytes.TNF-gamma-alpha and/or TNF-gamma-beta may also be employed to inhibitT-cell proliferation by the inhibition of IL-2 biosynthesis for thetreatment of T-cell mediated auto-immune diseases and lymphocyticleukemias. TNF-gamma-alpha and/or TNF-gamma-beta may also be employed tostimulate wound healing, both via the recruitment of debris clearing andconnective tissue promoting inflammatory cells. In this same manner,TNF-gamma-alpha and/or TNF-gamma-beta may also be employed to treatother fibrotic disorders, including liver cirrhosis, osteoarthritis andpulmonary fibrosis. TNF-gamma-alpha and/or TNF-gamma-beta also increasesthe presence of eosinophils which have the distinctive function ofkilling the larvae of parasites that invade tissues, as inschistosomiasis, trichinosis and ascariasis. TNF-gamma-alpha and/orTNF-gamma-beta may also be employed to regulate hematopoiesis, byregulating the activation and differentiation of various hematopoieticprogenitor cells, for example, to release mature leukocytes from thebone marrow following chemotherapy, i.e., in stem cell mobilization.TNF-gamma-alpha and/or TNF-gamma-beta may also be employed to treatsepsis.

In a similar fashion, TNF-gamma-alpha and/or TNF-gamma-beta may be usedto treat rheumatoid arthritis (RA) by inhibiting the increase inangiogensis or the increase in endothelial cell proliferation requiredto sustain an invading pannus in bone and cartilage as is often observedin RA. Endothelial cell proliferation is increased in the synovia of RApatients as compared to patients with osteoarthritis (OA) or unaffectedindividuals. Neovascularization is needed to sustain the increased massof the invading pannus into bone and cartilage. Inhibition ofangiogenesis is associated with a significant decrease in the severityof both early and chronic arthritis in animal models.

It will also be appreciated by one of ordinary skill that, since theTNF-gamma-alpha and/or TNF-gamma-beta proteins of the invention aremembers of the TNF family the mature secreted form of the protein may bereleased in soluble form from the cells which express TNF-gamma byproteolytic cleavage. Therefore, when TNF-gamma-alpha and/orTNF-gamma-beta mature form or soluble extracellular domain is added froman exogenous source to cells, tissues or the body of an individual, theprotein will exert its physiological activities on its target cells ofthat individual. Also, cells expressing this type II transmembraneprotein may be added to cells, tissues or the body of an individual andthese added cells will bind to cells expressing receptor forTNF-gamma-alpha and/or TNF-gamma-beta, whereby the cells expressingTNF-gamma-alpha and/or TNF-gamma-beta can cause actions (e.g. regulationof endothelial cell growth and regulation) on the receptor-bearingtarget cells.

Therefore, it will be appreciated that conditions caused by a decreasein the standard or normal level of TNF-gamma-alpha and/or TNF-gamma-betaactivities in an individual, particularly disorders of the immune andcirculatory systems, can be treated by administration of TNF-gamma-alphaand/or TNF-gamma-beta polypeptide (in the form of the mature protein).Thus, the invention also provides a method of treatment of an individualin need of an increased level of TNF-gamma-alpha and/or TNF-gamma-betaactivity comprising administering to such an individual a pharmaceuticalcomposition comprising an amount of an isolated TNF-gamma-alpha and/orTNF-gamma-beta polypeptide of the invention, particularly a mature formof the TNF-gamma-alpha and/or TNF-gamma-beta protein of the invention,effective to increase the TNF-gamma-alpha and/or TNF-gamma-beta activitylevel in such an individual.

The TNF-gamma-alpha and/or TNF-gamma-beta polypeptide of the presentinvention may be employed to inhibit tumor cell growth or neoplasia. TheTNF-gamma-alpha and/or TNF-gamma-beta polypeptide may be responsible fortumor destruction through apoptosis which is characterized by membraneblebbing (zeiosis), condensation of cytoplasma and the activation of anendogeneous endonuclease (FIG. 12). As shown in Table 1, TNF-gamma hasstrong cytotoxic activity for the cell lines tested which have abnormalcellular proliferation and regulation, for example the fibrosarcoma andcarcinoma cell line. This is also illustrated in FIGS. 7A, 7B, and 8where it is shown that TNF-gamma has the ability to inhibit L929 andWEHI 164 cell growth through cytotoxic activity. WEHI 164 cells aremouse fibrosarcoma cells. A preferable method of administering theTNF-gamma is by injection directly into the tumor.

The cell adhesion activity of TNF-gamma may be employed for woundhealing. As shown in Table 1 and FIG. 9, TNF-gamma has a strongendothelial cell proliferation effect which is an indication thatTNF-gamma plays a role in wound healing. TNF-gamma's cell adhesiveeffects may also play a role in wound healing.

TNF-gamma may also be employed to treat diseases which require growthpromotion activity, for example, restenosis. As stated above, TNF-gammais shown to have strong proliferation effects on endothelial cellgrowth. Accordingly, TNF-gamma may also be employed to regulatehematopoiesis and endothelial cell development.

The TNF-gamma polypeptide, through its ability to stimulate theactivation of T-cells, is an important mediator of the immune response.Accordingly, this polypeptide may be used to stimulate an immuneresponse against a variety of parasitic, bacterial and viral infections.TNF-gamma may lyse virus-infected cells and, therefore, be employed toarrest HIV infected cells.

The TNF-gamma polypeptide may also be employed to treat autoimmunediseases such as Type I diabetes by enhancing the T-cell proliferativeresponse.

TABLE 2 Summary of TNF-gamma activity Source Cyto- Prolif- Cell linesand type toxicity eration Differentiation Adhesion mouse L929fibroblast + − − − mouse WEHI 164 fibrosarcoma +++ − − − rat kidneyNRK-54E epithelial- + − − − like human THP-1 monocytic + − ++ ++leukemia human HL-60 promyelo- − − − ++ cytic leukemia human RajiBurkitt − − − − lymphoma human Jurkat T-cell ++ − − − lymphoma PrimaryHUVEC − ++ − ? human aterial Primary endothelial +* ++ − ? human A431epidermoid ++ − − − carcinoma human 293 embryonal − ++ − − kidneyLegend: *At high dose only. The numbers of “+” indicate the relativelevel of activities. “.” indicates no detected activity at theconcentration range tested.

TNF-gamma may be used to inhibit the proliferation of endothelial cells,for example, aortic endothelial cells. As illustrated in FIG. 10, atconcentrations of up to 10 μg/ml, TNF-gamma can nearly completelyinhibit the growth of endothelial cells while having no apparent effecton the growth of human breast cancer cells. As a result, TNF-gamma canbe used to treat diseases and disorders in which inhibition ofendothelial cell growth is advantageous. Inhibiting the growth ofendothelial cells is desirable in the treatment of many types of cancerswhich depend on the generation of new blood vessels to support growth ofthe tumor. TNF-gamma can be used to inhibit the growth of such tumors byinhibiting the growth of endothelial cells which are a major cellularcomponent of the blood vessel. Evidence of the ability of TNF-gamma tobe effectively used in this fashion is presented in FIGS. 16A and 16B.These experiments are discussed in greater detail below.

In particular, TNF-gamma can be used to regulate endothelial cell growthwhen endothelial cells have already begun proliferating. Such asituation may when angiogenesis is occurring as a tumor-supportingmechanism as described above. Endogenous TNF-gamma expression is reducedin proliferating cultures of endothelial cells, whereas the expressionof endogenous TNF-gamma is enhanced in quiescent endothelial cellcultures (FIG. 4). As a result, it is preferable to use TNF-gamma of thepresent invention to reduce the rate of cell growth in cultures ofproliferating endothelial cells, for example, during the increase insize of a tumor in a cancerous state.

TNF-gamma of the present invention has been used to reduce the formationof capillary-like tubular structures formed by endothelial cells invitro. As illustrated in FIG. 14, TNF-gamma of the present invention canbe used to inhibit the formation of endothelial cells organized intocapillary-like tubular structures in response to angiogenic factors suchas FGF-2. Furthermore, isolated TNF-gamma of the present invention canalso be used to inhibit the growth and organization of endothelial cellsinto capillary vessels in a modified chicken embryo chorioallantoicmembrane (CAM), as shown in FIG. 15. As a result, TNF-gamma of thepresent invention can be used to inhibit the formation of capillaries orcapillary-like structures from endothelial cells in vitro.

TNF-gamma of the present invention can be used as an anti-cancer agent.As illustrated in FIG. 16, TNF-gamma was used to inhibit the growth ofhuman breast cancer cells in a xenograft tumor model. Despite the hightumorigenicity of these cells, treatment with TNF-gamma of the presentinvention resulted in a marked inhibition of the growth of the xenografttumors. TNF-gamma, or a mutein thereof, of the present invention, can beused to treat a number of cancers including, but not limited to, breastcancer, colon cancer, cardiac tumors, pancreatic cancer, melanoma,retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicularcancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma,endothelioma, osteoblastoma, osteoclastoma, adenoma, and the like.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsand diagnostics to human disease.

This invention provides a method for identification of the receptor forTNF-gamma. The gene encoding the receptor can be identified by numerousmethods known to those of skill in the art, for example, ligand panningand FACS sorting (Coligan, et al., Current Protocols in Immun., 1(2),Chapter 5, (1991)). Preferably, expression cloning is employed whereinpolyadenylated RNA is prepared from a cell responsive to TNF-gamma, anda cDNA library created from this RNA is divided into pools and used totransfect COS cells or other cells that are not responsive to TNF-gamma.Transfected cells which are grown on glass slides are exposed to labeledTNF-gamma. TNF-gamma can be labeled by a variety of means includingiodination or inclusion of a recognition site for a site-specificprotein kinase. Following fixation and incubation, the slides aresubjected to autoradiographic analysis. Positive pools are identifiedand sub-pools are prepared and retransfected using an iterativesub-pooling and rescreening process, eventually yielding a single clonethat encodes the putative receptor.

As an alternative approach for receptor identification, labeledTNF-gamma can be photoaffinity-linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE and exposed to X-ray film. The labeled complexcontaining the TNF-gamma-receptor can be excised, resolved into peptidefragments, and subjected to protein microsequencing. The amino acidsequence obtained from microsequencing would be used to design a set ofdegenerate oligonucleotide probes to screen a cDNA library to identifythe gene encoding the putative receptor.

TNF-gamma does not bind significantly to two soluble TNF receptors,sTNF-RI (p55) and sTNF-RII (p75). Accordingly, TNF-gamma may haveactivities inclusive of and additional to known TNF proteins (see FIG.13).

Formulations

The TNF-gamma polypeptide composition will be formulated and dosed in afashion consistent with good medical practice, taking into account theclinical condition of, the individual patient (especially the sideeffects of treatment with TNF-gamma polypeptide alone), the site ofdelivery of the TNF-gamma polypeptide composition, the method ofadministration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” of TNF-gamma polypeptidefor purposes herein is thus determined by such considerations.

The antagonists may be employed in a composition with a pharmaceuticallyacceptable carrier, e.g., as hereinafter described.

The TNF-gamma polypeptides and agonists and antagonists of the presentinvention may be employed in combination with a suitable pharmaceuticalcarrier. Such compositions comprise a therapeutically effective amountof the compound, and a pharmaceutically acceptable carrier or excipient.Such a carrier includes but is not limited to saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Theformulation should suit the mode of administration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepharmaceutical compositions of the present invention may be employed inconjunction with other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmanner such as by the topical, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes. Thepharmaceutical compositions are administered in an amount which iseffective for treating and/or prophylaxis of the specific indication. Ingeneral, they are administered in an amount of at least about 10 g/kgbody weight and in most cases they will be administered in an amount notin excess of about 8 mg/Kg body weight per day. In most cases, thedosage is from about 10 g/kg to about 1 mg/kg body weight daily, takinginto account the routes of administration, symptoms, etc.

Gene Therapy

The TNF-gamma polypeptides and agonists and antagonists which arepolypeptides may also be employed in accordance with the presentinvention by expression of such polypeptides in vivo, which is oftenreferred to as “gene therapy.”

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art and are apparentfrom the teachings herein. For example, cells may be engineered by theuse of a retroviral particle containing RNA encoding a polypeptide ofthe present invention.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Forexample, a producer cell for producing a retroviral particle containingRNA encoding a polypeptide of the present invention may be administeredto a patient for engineering cells in vivo and expression of thepolypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

Retroviruses from which the retroviral plasmid vectors hereinabovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

The vector includes one or more promoters. Suitable promoters which maybe employed include, but are not limited to, the retroviral LTR; theSV40 promoter; and the human cytomegalovirus (CMV) promoter described byMiller and colleagues (Biotechniques 7:980-990 (1989)), or any otherpromoter (e.g., cellular promoters such as eukaryotic cellular promotersincluding, but not limited to, the histone, pol III, and b-actinpromoters). Other viral promoters which may be employed include, but arenot limited to, adenovirus promoters, thymidine kinase (TK) promoters,and B19 parvovirus promoters. The selection of a suitable promoter willbe apparent to those skilled in the art from the teachings containedherein.

The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthyridine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the, modified retroviral LTRshereinabove described); the bactin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, b-2,b-AM, PA12, T19-14X, VT-19-17-H2, CRE, b-CRIP, GP+E-86, GP+envAm12, andDAN cell lines as described by Miller (Human Gene Therapy 1:5-14(1990)), which is incorporated herein by reference in its entirety. Thevector may transduce the packaging cells through any means known in theart. Such means include, but are not limited to, electroporation, theuse of liposomes, and CaPO₄ precipitation. In one alternative, theretroviral plasmid vector may be encapsulated into a liposome, orcoupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the polypeptides.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

Agonists and Antagonists—Assays and Molecules

This invention is also related to a method of screening compounds toidentify those which mimic TNF-gamma (agonists) or prevent the effect ofTNF-gamma (antagonists). An example of such a method takes advantage ofthe ability of TNF-gamma to significantly stimulate the proliferation ofhuman endothelial cells in the presence of the comitogen Con A.Endothelial cells are obtained and cultured in 96-well flat-bottomedculture plates (Costar, Cambridge, Mass.) in RPMI 1640 supplemented with10% heat-inactivated fetal bovine serum (Hyclone Labs, Logan, Utah), 1%L-glutamine, 100 U/ml penicillin, 100 g/ml steptomycin, 0.1% gentamycin(Gibco Life Technologies, Grand Island, N.Y.) in the presence of 2 g/mlof Con-A (Calbiochem, La Jolla, Calif.). Con-A, and the compound to bescreened are added to a final volume of 0.2 ml. After 60 h at 37° C.,cultures are pulsed with 1 Ci of [³H]thymidine (5 Ci/mmol; 1 Ci=37 BGq;NEN); for 12-18 h and harvested onto glass fiber filters (PhD; CambridgeTechnology, Watertown, Mass.). Mean [³H]thymidine incorporation (cpm) oftriplicate cultures is determined using a liquid scintillation counter(Beckman Instruments, Irvine, Calif.). Significant [³H]-thymidineincorporation indicates stimulation of endothelial cell proliferation.

Alternatively, the response of a known second messenger system followinginteraction of TNF-gamma and receptor would be measured and compared inthe presence or absence of the compound. Such second messenger systemsinclude but are not limited to, cAMP guanylate cyclase, ion channels orphosphoinositide hydrolysis.

To assay for antagonists, the assay described above is performed,however, in this assay TNF-gamma is added along with the compound to bescreened and the ability of the compound to inhibit [³H]thymidineincorporation in the presence of TNF-gamma, indicates that the compoundis an antagonist to TNF-gamma. Alternatively, TNF-gamma antagonists maybe detected by combining TNF-gamma and a potential antagonist withmembrane-bound TNF-gamma receptors or recombinant receptors underappropriate conditions for a competitive inhibition assay. TNF-gamma canbe labeled, such as by radioactivity, such that the number of TNF-gammamolecules bound to the receptor can determine the effectiveness of thepotential antagonist.

Alternatively, a mammalian cell or membrane preparation expressing theTNF-gamma receptor is incubated with labeled TNF-gamma in the presenceof the compound. The ability of the compound to enhance or block thisinteraction could then be measured.

In another aspect of this embodiment the invention provides a method foridentifying a receptor protein or other ligand-binding protein whichbinds specifically to a TNF-gamma polypeptide (e.g. DR3). For example, acellular compartment, such as a membrane or a preparation thereof, maybe prepared from a cell that expresses a molecule that binds TNF-gamma.The preparation is incubated with labeled TNF-gamma and complexes ofTNF-gamma bound to the receptor or other binding protein are isolatedand characterized according to routine methods known in the art.Alternatively, the TNF-gamma polypeptide may be bound to a solid supportso that binding molecules solubilized from cells are bound to the columnand then eluted and characterized according to routine methods.

In the assay of the invention for agonists or antagonists, a cellularcompartment, such as a membrane or a preparation thereof, may beprepared from a cell that expresses a molecule that binds TNF-gamma,such as a molecule of a signaling or regulatory pathway modulated byTNF-gamma. The preparation is incubated with labeled TNF-gamma in theabsence or the presence of a candidate molecule which may be a TNF-gammaagonist or antagonist. The ability of the candidate molecule to bind thebinding molecule is reflected in decreased binding of the labeledligand. Molecules which bind gratuitously, i.e., without inducing theeffects of TNF-gamma on binding the TNF-gamma binding molecule, are mostlikely to be good antagonists. Molecules that bind well and eliciteffects that are the same as or closely related to TNF-gamma areagonists.

TNF-gamma-like effects of potential agonists and antagonists may bymeasured, for instance, by determining activity of a second messengersystem following interaction of the candidate molecule with a cell orappropriate cell preparation, and comparing the effect with that ofTNF-gamma or molecules that elicit the same effects as TNF-gamma. Secondmessenger systems that may be useful in this regard include but are notlimited to AMP guanylate cyclase, ion channel or phosphoinositidehydrolysis second messenger systems.

Another example of an assay for TNF-gamma antagonists is a competitiveassay that combines TNF-gamma and a potential antagonist withmembrane-bound TNF-gamma receptor molecules or recombinant TNF-gammareceptor molecules under appropriate conditions for a competitiveinhibition assay. TNF-gamma can be labeled, such as by radioactivity,such that the number of TNF-gamma molecules bound to a receptor moleculecan be determined accurately to assess the effectiveness of thepotential antagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polypeptide of the inventionand thereby inhibit or extinguish its activity. Potential antagonistsalso may be small organic molecules, a peptide, a polypeptide such as aclosely related protein or antibody that binds the same sites on abinding molecule, such as a receptor molecule, without inducingTNF-gamma-induced activities, thereby preventing the action of TNF-gammaby excluding TNF-gamma from binding.

Other potential antagonists include antisense molecules. Antisensetechnology can be used to control gene expression through antisense DNAor RNA or through triple-helix formation. Antisense techniques arediscussed in a number of studies (for example, Okano, J. Neurochem.56:560 (1991); “Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression.” CRC Press, Boca Raton, Fla. (1988)). Triple helix formationis discussed in a number of studies, as well (for instance, Lee, et al.,Nucleic Acids Research 6:3073 (1979); Cooney, et al., Science 241:456(1988); Dervan, et al., Science 251:1360 (1991)). The methods are basedon binding of a polynucleotide to a complementary DNA or RNA. Forexample, the 5′ coding portion of a polynucleotide that encodes themature polypeptide of the present invention may be used to design anantisense RNA oligonucleotide of from about 10 to 40 base pairs inlength. A DNA oligonucleotide is designed to be complementary to aregion of the gene involved in transcription thereby preventingtranscription and the production of TNF-gamma. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into TNF-gamma polypeptide. The oligonucleotidesdescribed above can also be delivered to cells such that the antisenseRNA or DNA may be expressed in vivo to inhibit production of TNF-gammaprotein.

Antibodies specific to TNF-gamma may be used as antagonists by bindingto TNF-gamma and preventing it from binding to its receptor. Monoclonalantibodies are particularly effective in this regard. Antibodiesspecific to the TNF-gamma receptor, however, may mediate distinctcellular responses which tend to agonize the effects of TNF-gamma uponinteraction with its receptor.

Potential TNF-gamma antagonists also include TNF-gamma mutants whichbind to the TNF-gamma receptor and elicit no second messenger responseto effectively block the receptor from its natural ligand. Specificallydesigned oligonucleotides and small molecules may also bind to theTNF-gamma receptor (e.g., DR3) and block it from TNF-gamma. Examples ofsmall molecules include but are not limited to small peptides orpeptide-like molecules.

Another potential TNF-gamma antagonist is a soluble form of theTNF-gamma receptor which binds to TNF-gamma and prevents it frominteracting with membrane-bound TNF-gamma receptors. In this way, thereceptors are not stimulated by TNF-gamma.

Another potential TNF-gamma antagonist is an antisense constructprepared using antisense technology. Antisense technology can be used tocontrol gene expression through triple-helix formation or antisense DNAor RNA, both of which methods are based on binding of a polynucleotideto DNA or RNA. For example, the 5′ coding portion of the polynucleotidesequence, which encodes for the mature polypeptides of the presentinvention, is used to design an antisense RNA oligonucleotide of fromabout 10 to 40 base pairs in length. A DNA oligonucleotide is designedto be complementary to a region of the gene involved in transcription(triple helix—see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney etal, Science, 241:456 (1988); and Dervan et al., Science, 251: 1360(1991)), thereby preventing transcription and the production ofTNF-gamma. The antisense RNA oligonucleotide hybridizes to the mRNA invivo and blocks translation of the mRNA molecule into the TNF-gammapolypeptide (Antisense—Okano, J. Neurochem., 56:560 (1991);Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)). The oligonucleotides described abovecan also be delivered to cells such that the antisense RNA or DNA may beexpressed in vivo to inhibit production of TNF-gamma.

TNF-antagonists may also be employed to treat cachexia which is a lipidclearing defect resulting from a systemic deficiency of lipoproteinlipase which is suppressed by TNF-gamma. The TNF-gamma antagonists arealso employed to treat cerebral malaria in which TNF-gamma appears toplay a pathogenic role. The antagonists may also be employed to treatrheumatoid arthritis by inhibiting TNF-gamma induced production ofinflammatory cytokines such as IL-1 in the synovial cells. When treatingarthritis TNF-gamma is preferably injected intra-articularly.

The TNF-gamma antagonists may also be employed to prevent graftrejection by preventing the stimulation of the immune system in thepresence of a graft by TNF-gamma.

The TNF-gamma antagonists may also be employed to treat osteoporosissince TNF-gamma may induce bone resorption.

Antagonists to TNF-gamma may also be employed as anti-inflammationagents since TNF-gamma mediates an enhanced inflammatory response.

The antagonists may also be used to treat endotoxic shock, also referredto as septic shock. This critical condition results from an exaggeratedresponse to bacterial or other types of infection. This response leadsto elevated levels of TNF-gamma which causes shock and tissue injury.

The present invention also relates to a diagnostic assay for detectingaltered levels of TNF-gamma protein in various tissues since anover-expression of the proteins compared to normal control tissuesamples may detect the presence of a disease or susceptibility to adisease, for example, tumors and cerebral malaria. Assays used to detectlevels of TNF-gamma protein in a sample derived from a host arewell-known to those of skill in the art and include radioimmunoassays,competitive-binding assays, Western Blot analysis, ELISA assays and“sandwich” assay. An ELISA assay (Coligan, et al., Current Protocols inImmunology, 1(2), Chapter 6, (1991)) initially comprises preparing anantibody specific to the TNF-gamma antigen, preferably a monoclonalantibody. In addition a reporter antibody is prepared against themonoclonal antibody. To the reporter antibody is attached a detectablereagent such as radioactivity, fluorescence or in this example ahorseradish peroxidase enzyme. A sample is removed from a host andincubated on a solid support, e.g. a polystyrene dish, that binds theproteins in the sample. Any free protein binding sites on the dish arethen covered by incubating with a non-specific protein like BSA. Next,the monoclonal antibody is incubated in the dish during which time themonoclonal antibodies attach to any TNF-gamma proteins attached to thepolystyrene dish. All unbound monoclonal antibody is washed out withbuffer. The reporter antibody linked to horseradish perosxidase is nowplaced in the dish resulting in binding of the reporter antibody to anymonoclonal antibody bound to TNF-gamma. Unattached reporter antibody isthen washed out. Peroxidase substrates are then added to the dish andthe amount of color developed in a given time period is a measurement ofthe amount of TNF-gamma protein present in a given volume of patientsample when compared against a standard curve.

A competition assay may be employed wherein antibodies specific toTNF-gamma are attached to a solid support and labeled TNF-gamma and asample derived from the host are passed over the solid support and theamount of label detected, for example by liquid scintillationchromotagraphy, can be correlated to a quantity of TNF-gamma in thesample.

A “sandwich” assay is similar to an ELISA assay. In a “sandwich” assayTNF-gamma is passed over a solid support and binds to antibody attachedto a solid support. A second antibody is then bound to the TNF-gamma. Athird antibody which is labeled and specific to the second antibody isthen passed over the solid support and binds to the second antibody andan amount can then be quantitated.

Gene Mapping

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region of the sequence is used to rapidly select primersthat do not span more than one exon in the genomic DNA, thuscomplicating the amplification process. These primers are then used forPCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with cDNA as short as 50 or 60bases. For a review of this technique, see Verma et al., HumanChromosomes: a Manual of Basic Techniques, Pergamon Press, New York(1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

Utilizing the techniques described above, the chromosomal location ofTNF-gamma was determined with very high confidence to be 9q32. Previouschromosomal mapping studies have linked several developmental defects toloci in this area of chromosome 9.

EXAMPLES

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples certainfrequently occurring methods and/or terms will be described.

“Plasmids” are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures, unless otherwise stated. In addition, equivalent plasmids tothose described are known in the art and will be apparent to theordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 g of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37 C are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,8:4057 (1980).

“Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units of T4 DNA ligase (“ligase”)per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

Unless otherwise stated, transformation was performed as described inthe method of Graham, F. and Van der Eb, A., Virology, 52:456-457(1973).

Example 1 Bacterial Expression and Purification of TNF-Gamma

The DNA sequence encoding the full-length TNF-gamma ORF, ATCC DepositNo. 75927, was initially amplified using PCR oligonucleotide primerscorresponding to the 5′ and 3′ sequences of the TNF-gamma protein.Additional nucleotides corresponding to TNF-gamma were added to the 5′and 3′ sequences respectively. The 5′ oligonucleotide primer is shown asSEQ ID NO:13 and has the sequence 5′-GCG CGG ATC CAC CAT GAG ACG CTT TTTAAG CAA AGT C-3′ which contains a Bam HI restriction enzyme sitefollowed by the first 24 nucleotides of TNF-gamma coding sequencestarting from the initiating methionine codon. The 3′ sequence 5′-CGCGTC TAG ACT ATA GTA AGA AGG CTC CAA AGA AGG-3′ (SEQ ID NO:14) containssequences complementary to an Xba I site and 22 nucleotides ofTNF-gamma. The restriction enzyme sites correspond to the restrictionenzyme sites in the bacterial expression vector pQE-9 (Qiagen). pQE-9was then digested with Bam HI and Xba I. The amplified sequences wereligated into pQE-9 and were inserted in frame with the sequence encodingfor the histidine tag and the RBS. The ligation mixture was then used totransform an E. coli strain available from Qiagen under the trademarkM15/rep 4 by the procedure described in Sambrook, J. et al., MolecularCloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989).M15/rep4 contains multiple copies of the plasmid pREP4, which expressesthe lacI repressor and also confers kanamycin resistance (Kan^(r)).Transformants were identified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies were selected. Plasmid DNA wasisolated and confirmed by restriction analysis. Clones containing thedesired constructs were grown overnight (O/N) in liquid culture in LBmedia supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/Nculture was used to inoculate a large culture at a ratio of 1:100 to1:250. The cells were grown to an optical density 600 (O.D.₆₀₀) ofbetween 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalactopyranoside”) wasthen added to a final concentration of 1 mM. IPTG induces byinactivating the lacI repressor, clearing the P/O leading to increasedgene expression. Cells were grown an extra 3 to 4 hours. Cells were thenharvested by centrifugation. The cell pellet was solubilized in thechaotropic agent 6 M Guanidine HCl (Guanidine HCl concentrations ofgreater than or equal to 2.5 M were empirically found to resulat in ahigher level of purity of recovered recombinant protein). Afterclarification, solubilized TNF-gamma was purified from this solution bychromatography on a Nickel-Chelate column under conditions that allowfor tight binding by proteins containing the 6-His tag (Hochuli, E. etal., J. Chromatography 411:177-184 (1984)). TNF-gamma was furtherpurified by a second run on the Nickel-chelate column. TNF-gamma (90%pure) was eluted from the column in 6 M guanidine HCl pH 5.0 and for thepurpose of renaturation was dialyzed in PBS buffer. The expressionproduct was electrophoresed by SDS-PAGE, and the results may be seen inFIG. 5 where lanes labeled “M” contain molecular weight markers; lane 1is induced cell lysate; lane 2 is uninduced call lysate; lane 3 is theTNF-gamma protein after two Nickel-chelate column purifications; lane 4is the TNF-gamma protein after 1 column purification.

One of ordinary skill in the art will recognize that bacterialexpression vectors other than pQE-9 may also be used to expressTNF-gamma. One such preferred bacterial expression vector is pHE4-5.pHE4-5 may be obtained as pHE4-5/MPIFD23 plasmid DNA (this constructcontains an unrelated insert which encodes an unrelated ORF). ThepHE4-5/MPIFD23 plasmid was deposited with the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209, onSep. 30, 1997 (Accession No. 209311). Using the Nde I and Asp 718restriction sites flanking the unrelated MPIF ORF insert, one ofordinary skill in the art could easily use current molecular biologicaltechniques to replace the unrelated ORF in the pHE4-5/MPIFD23 plasmidwith the TNF-gamma ORF, or variations thereof, of the present invention.

Example 2 Cloning and Expression of TNF-Gamma Using the BaculovirusExpression System

The DNA sequence encoding the full length TNF-gamma protein, ATCC No.75927, was amplified using PCR oligonucleotide primers corresponding tothe 5′ and 3′ sequences of the gene: The 5′ primer has the sequence5′-GCG CGG ATC CAC CAT GAG ACG CTT TTT AAG CAA AGT C-3′ (SEQ ID NO:15)and contains a Bam HI restriction enzyme site (in bold) followed by 24nucleotides of the TNF-gamma gene (the initiation codon for translation“ATG” is underlined). The 3′ primer has the sequence 5′-CGC GTC TAG ACTATA GTA AGA AGG CTC CAA AGA AGG-3′ (SEQ ID NO:16) and contains thecleavage site for the restriction endonuclease Xba I and 22 nucleotidescomplementary to the 3′ non-translated sequence of the TNF-gamma gene.The amplified sequences were isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Ca.).The fragment was then digested with the endonucleases Bam HI and Xba Iand then purified again on a 1% agarose gel. This fragment wasdesignated F2.

The vector pA2 (modification of pVL941 vector, discussed below) was usedfor the expression of the TNF-gamma protein using the baculovirusexpression system (for review see: Summers, M. D. and Smith, G. E. 1987,A manual of methods for baculovirus vectors and insect cell cultureprocedures, Texas Agricultural Experimental Station Bulletin No. 1555).This expression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed bythe recognition sites for the restriction endonucleases Bam HI and XbaI. The polyadenylation site of the simian virus SV40 is used forefficient polyadenylation. For an easy selection of recombinant virusthe beta-galactosidase gene from E. coli was inserted in the sameorientation as the polyhedrin promoter followed by the polyadenylationsignal of the polyhedrin gene. The polyhedrin sequences were flanked atboth sides by viral sequences for the cell-mediated homologousrecombination of cotransfected wild-type viral DNA. Many otherbaculovirus vectors could have been used in place of pA2, such as pRG1,pAc373, pVL941 and pAcIM1 (Luckow, V. A. and Summers, M. D., Virology,170:31-39).

The plasmid was digested with the restriction enzymes Bam HI and Xba Iand then dephosphorylated using calf intestinal phosphatase byprocedures known in the art. The DNA was then isolated from a 1% agarosegel using the commercially available kit (“Geneclean” BIO 101 Inc., LaJolla, Ca.). This vector DNA was designated V2.

Fragment F2 and the dephosphorylated plasmid V2 were ligated with T4 DNAligase. E. coli XL1 blue cells were then transformed. The sequence ofthe cloned fragment was confirmed by DNA sequencing.

5 μg of the plasmid pBac TNF-gamma was cotransfected with 1.0 μg of acommercially available linearized baculovirus (“BaculoGold baculovirusDNA”, Pharmingen, San Diego, Calif.) using the lipofection method(Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

1μg of BaculoGold virus DNA and 5 μg of the plasmid pBac TNF-gamma weremixed in a sterile well of a microtiter plate containing 50 μl of serumfree Grace's medium (Life Technologies Inc., Gaithersburg, Md.).Afterwards, 10 μl Lipofectin plus 90 μl Grace's medium were added, mixedand incubated for 15 minutes at room temperature. Then the transfectionmixture was added dropwise to the Sf9 insect cells (ATCC CRL 1711)seeded in a 35 mm tissue culture plate with 1 ml Grace' medium withoutserum. The plate was rocked back and forth to mix the newly addedsolution. The plate was then incubated for 5 hours at 27° C. After 5hours, the transfection solution was removed from the plate and 1 ml ofGrace's insect medium supplemented with 10% fetal calf serum was added.The plate was put back into an incubator and cultivation continued at27° C. for four days.

After four days, the supernatant was collected and a plaque assayperformed essentially as described by Summers and Smith (supra). As amodification, an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) was used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

Four days after the serial dilution, the virus was added to the cells,blue stained plaques were picked with the tip of an Eppendorf pipette.The agar containing the recombinant viruses was then resuspended in anEppendorf tube containing 200 μl of Grace's medium. The agar was removedby a brief centrifugation and the supernatant containing the recombinantbaculovirus was used to infect Sf9 cells seeded in 35 mm dishes. Fourdays later the supernatants of these culture dishes were harvested andthen stored at 4° C.

Sf9 cells were grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells were infected with the recombinantbaculovirus V-TNF-gamma at a multiplicity of infection (MOI) of 2. Sixhours later the medium was removed and replaced with SF900 II mediumminus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42hours later 5 μCi of [³⁵S]-methionine and 5 μCi [³⁵S]cysteine (Amersham)were added. The cells were further incubated for 16 hours before theywere harvested by centrifugation and the labeled proteins visualized bySDS-PAGE and autoradiography. FIG. 6 illustrates a gel where lanes 1 and3 are the medium of the TNF-gamma and control cultures and lanes 2 and 4are the cell lysates of the TNF-gamma and the control cultures.

Example 3 Expression of Recombinant TNF-Gamma in COS Cells

The expression of plasmid, TNF-gamma-HA is derived from a vectorpcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2)ampicillin resistance gene, 3) E. coli replication origin, 4) CMVpromoter followed by a polylinker region, an SV40 intron, and apolyadenylation site. A DNA fragment encoding the entire TNF-gammaprecursor and a hemagglutinin antigen (HA) tag fused in frame to its 3′end was cloned into the polylinker region of the vector. Therefore, therecombinant protein expression is under the direction of the CMVpromoter. The HA tag corresponds to an epitope derived from theinfluenza hemagglutinin protein as previously described (1. Wilson, H.Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell37, 767). The fusion of HA tag to our target protein allows easydetection of the recombinant protein with an antibody that recognizesthe HA epitope.

The plasmid construction strategy is described as follows: The DNAsequence encoding TNF-gamma, ATCC #75927, was constructed by PCR on theoriginal EST cloned using two primers: the 5′ primer (SEQ ID NO:15)contains a Bam HI site followed by 24 nucleotides of TNF-gamma codingsequence starting from the initiation codon; the 3′ sequence 5′-CGC TCTAGA TCA AGC GTA GTC TGG GAC GTC GTA TGG ATA GTA AGA AGG CTC CAA AG-3′(SEQ ID NO:17) contains complementary sequences to Xba I site,translation stop codon, HA tag and the last 18 nucleotides of theTNF-gamma coding sequence (not including the stop codon). Therefore, thePCR product contained a Bam HI site, TNF-gamma coding sequence followedby HA tag fused in frame, a translation termination stop codon next tothe HA tag, and an Xba I site. The PCR amplified DNA fragment and thevector, pcDNAI/Amp, were digested with Bam HI and Xba I restrictionenzymes and ligated together. The ligation mixture was transformed intoE. coli strain SURE (available from Stratagene Cloning Systems, 11099North Torrey Pines Road, La Jolla, Calif. 92037) the transformed culturewas plated on ampicillin media plates and resistant colonies wereselected. Plasmid DNA was isolated from transformants and examined byrestriction analysis for the presence of the correct fragment. Forexpression of the recombinant TNF-gamma, COS cells were transfected withthe expression vector by DEAE-DEXTRAN method. (J. Sambrook, E. Fritsch,T. Maniatis, Molecular Cloning. A Laboratory Manual, Cold SpringLaboratory Press, (1989)). The expression of the TNF-gamma HA proteinwas detected by radiolabelling and immunoprecipitation method. (E.Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, (1988)). Cells were labeled for 8 hours with[³⁵S]-S-cysteine two days post transfection. Culture media were thencollected and cells were lysed with detergent (RIPA buffer (150 mM NaCl,1% NP-40, 0.1% SDS, 1% NP40, 0.5% DOC, 50 mM Tris, pH 7.5; Wilson, I. etal., Id. 37:767 (1984)). Both cell lysate and culture media wereprecipitated with an HA-specific monoclonal antibody. Precipitatedproteins were then analyzed on 15% SDS-PAGE gels.

Example 4 Expression Pattern of TNF-Gamma in Human Tissue

RNA blot analysis was carried out to examine the levels of expression ofTNF-gamma in human tissues. Total cellular RNA samples were isolatedwith RNAzol™ B system (Biotecx Laboratories, Inc. 6023 South Loop East,Houston, Tex. 77033). About 2 μg (for the RNA blot of FIG. 3A) of totalRNA isolated from each human tissue specified was separated on 1%agarose-formaldehyde gel and blotted onto a nylon filter (Sambrook,Fritsch, and Maniatis, Molecular Cloning, Cold Spring Harbor Press,(1989)). The labeling reaction was done according to the StratagenePrime-It kit with 50 ng TNF-gamma cDNA, to produce [³²P]-labeledTNF-gamma cDNA. The labeled DNA was purified with a Select-G-50 column(5 Prime—3 Prime, Inc. 5603 Arapahoe Road, Boulder, Colo. 80303). Thefilter was then hybridized with radioactive labeled full-lengthTNF-gamma gene at 1,000,000 cpm/ml in 0.5 M NaPO₄, pH 7.4 and 7% SDSovernight at 65° C. After being washed twice at room temperature andtwice at 60° C. with 0.5×SSC, 0.1% SDS, the X-ray film was then exposedto the blot at −70° C. overnight with an intensifying screen. Themessage RNA for TNF-gamma is abundant in kidney.

The same reaction was done to obtain the results shown in FIG. 3B, withthe exception that 10 μg poly-A RNA isolated from the indicated tissueswas used. In this experiment, the messenger RNA encoding TNF-gamma isexpressed predominantly in HUVEC cells (FIG. 3B; lane 9), but not inother cell lines examined; for example; lane 1 is CAMA1 (breast cancer);lane 2 is AN3CA (uterine cancer); lane 3 is SK.UT.1 (uterine cancer);lane 4 is MG63 (osteoblastoma); lane 5 is HOS (osteoblastoma); lane 6 isMCF7 (breast cancer); lane 7 is OVCAR-3 (ovarian cancer); lane 8 isCAOV-3 (ovarian cancer); lane 10 is AOSMIC (smooth muscle); and lane 11is foreskin fibroblast.

Northern blot analyses were also performed to determine the relativeexpression level of the TNF-gamma RNA with respect to the proliferationstate of HUVEC cell cultures. In these experiments, identical amounts oftotal RNA isolated from HUVEC cells (15 μg) were electrophoreticallyseparated and blotted as described above. RNA was isolated from cultures1, 2, 3, 4, 6, and 7 days post-seeding. As illustrated in FIG. 4,TNF-gamma RNA (labeled “VEGI”) was only seen at low levels in newlyseeded cultures (days 1, 2, and 3). However, expression of TNF-gamma RNAwas apparent as the HUVEC cells in the cultures began to reachconfluence (days 4, 6, and 7). These experiments indicate that TNF-gammaexpression increases as cells in a culture or tissue begin to reach thequiescent state of non- or reduced-proliferation.

Example 5 Ability of Recombinant TNF-Gamma to Inhibit WEHI 164, ABAE,and L929 Cell Growth, and to Induce Cell Adhesion in HL-60 Cells

The adherent target cells were prepared from confluent cultures bytrypsinization in PBS, and non-adherent target cells were harvested fromstationary cultures and washed once with medium. Target cells weresuspended at 3×10⁵ cells/ml in medium containing 10% FCS. 0.1 mlaliquots were dispensed into 96-well flat-bottomed microtiter platescontaining 0.1 ml serially diluted test samples of cells (WEHI 164 andL929). Incubation was continued for 70 hours. TNF-a, TNF-b andbacterially-produced TNF-gamma were added at a 0.5 μg/ml concentration.The cytotoxicity and proliferation activity was quantified using an MTSassay performed by the addition of 20 μl of MTS and phenazinemethosulfate (PMS) solution to each well. After a three hour incubation,the OD at 492 nm was measured by an ELISA plate reader. The OD₄₉₂ isproportional to the number of viable cells in the wells. The percent ofcytotoxicity was calculated as follows: %cytotoxicity=(100−OD_(experimental)/OD_(control))×100. The photographswere taken after 72 hours. As shown by FIGS. 7A and 8, TNF-gamma induceda morphology change which appeared as dark round cells (indicatingkilled cells).

In the graph of FIG. 7B, the assay was performed as described above,however, increasing amounts of TNF-a, TNF-b and TNF-gamma were added tothe cultures. The results indicate that TNF-gamma is a dose-dependentinhibitor of the growth of the endothelial cell line WEHI 164, but notof the fibroblast cell line L929 (FIGS. 8 and 9).

A truncated form of the TNF-gamma polypeptide consisting of amino acids12-147 of the complete TNF-gamma amino acid sequence shown as SEQ IDNO:2 (designated TNF-gamma₁₂₋₁₄₇) was also used to examine the effect ofTNF-gamma on endothelial cell growth. Treatment of adult bovine aorticendothelial (ABAE) cells with TNF-gamma₁₂₋₁₄₇ resulted in nearlycomplete inhibition of the growth of cells in the ABAE culture, but notof cells in the breast cancer cell lines MDA-MB-435 or MDA-MB-231 (FIG.10; TNF-gamma is designated “VEGI” in this figure). Nearly completeinhibition of the growth of the endothelial cells was achieved at 10μg/ml TNF-gamma₃₉₋₁₇₄, with a half-maximum inhibitory concentrationvalue (IC₅₀) of approximately 1 μg/ml (approximately 70 nM).

To test adhesion ability of TNF-gamma, HL-60 cells were used and celladhesion and cell-cell contact were measured by observation under themicroscope and scored subjectively by two independent investigators.FIG. 11 illustrates the ability of TNF-gamma to induce cell adhesion.Cultures which were not treated with TNF-gamma contained cells which hadspread throughout the culture dish. However, cultures which were treatedwith TNF-gamma, contained cells which were clearly aggregated together.

Example 6 Measurement of Apoptosis Ability of TNF-Gamma

In a first incubation step, anti-histone antibody was fixed adsorptivelyon the wall of a microtiter plate module. Subsequently, non-specificbinding sites on the wall were saturated by treatment with incubationbuffer (e.g., blocking solution). During the second incubation step, thenucleosomes contained in the WEHI 164 cell sample treated with theTNF-a, TNF-b or bacterially-produced TNF-gamma were bound via theirhistone components to the immobilized anti-histone antibody. In thethird incubation step, anti-DNA-peroxidase (POD) reacted with the DNAcomponent of the nucleosomes. After removal of all unbound peroxidaseconjugate by a washing step, the amount of peroxidase retained in theimmunocomplex was determined spectrophotometrically using the substrateABTS (2,2′-azino-di-[3-ethylbenzthiazoline sulfonate]). Anti-histoneantibody reacted with the histones H1, H2A, H2B, H3, and H4 from thesample. Anti-DNA POD antibody bound to single- and double-stranded DNA.Therefore, the ELISA allowed the detection of mono- and oligonucleosomesand may be applied to measure apoptotic cell death. The level of celldeath was measured by the amount of cytoplasmic histone-associated DNAfragments which was indicated by the ratio of the absorbances observedat 405 and 490 nm (A₄₀₅/A₄₉₀). The results of these experiments areillustrated in FIG. 12 (See Boehringer mannheim Catalogue, 0990 C 93 21541170).

As shown in FIG. 12, WEHI 164 cells were induced to undergo increasinglyhigh levels of apoptosis, resulting in cell death, in the presence ofincreasing amounts of TNF-gamma. This effect was also observed in thepresence of increasing amounts of the control TNF-b or in the presenceof any of the analyzed levels of the control TNF-a.

Example 7 Receptor Binding Assay Using TNF-Gamma

TNF-a and bacterially-produced TNF-gamma were purified by Ni-NTAaffinity chromatography using the 6-His tag fused to the terminus of therecombinant proteins. 1 μg/well of either protein was added to a nickelchelate-coated 96-well plate (Xenopore Corp.) and incubated for 2 hours.After washing three times, 100 ng of human soluble TNF receptors(specifically, sTNF RI or sTNF RII) was added to each well and incubatedfor 2 hours. The plate was then washed three times and alkalinephosphatase-labeled polyclonal antibodies raised against either sTNF RIor sTNF RII was added in a total volume of 200 μl. An aliquot ofsubstrate solution (200 μl) was then added to each well and the platewas incubated for an additional 2 hours. The OD was then measured usingan ELISA reader (at a test wavelength of 450 nm and a correctionwavelength of 590 nm). The results shown in FIG. 13 illustrate thatTNF-gamma does not bind significantly to sTNF-receptors when compared tothe control binding observed with TNF-a.

Example 8 Expression via Gene Therapy

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask. At this time, fresh media is added (e.g., Ham's F12 media,supplemented with 10% FBS, penicillin, and streptomycin). The culture isthen incubated at 37° C. for approximately one week. At this time, freshmedia is added and subsequently changed every 2-3 days. After anadditional two weeks in culture, a monolayer of fibroblasts will haveemerged. The: monolayer is trypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988)), which is flankedby the long terminal repeats of the Moloney murine sarcoma virus, isdigested with Eco RI and Hind III, and, subsequently, treated with calfintestinal phosphatase. The linear vector is fractionated on agarose geland purified using glass beads.

The cDNA encoding a polypeptide of the present invention is amplifiedusing PCR primers which correspond to the 5′ and 3′ end sequencesrespectively. The 5′ primer containing an Eco RI site and the 3′ primerincludes a Hind III site. Equal quantities of the Moloney murine sarcomavirus linear backbone and the amplified Eco RI and Hind III fragment areadded together, in the presence of T4 DNA ligase. The resulting mixtureis maintained under conditions appropriate for ligation of the twofragments. The ligation mixture is used to transform bacteria HB 101,which are then plated onto agar-containing kanamycin for the purpose ofconfirming that the vector had the gene of interest properly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellsare transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

Fresh media is added to the transduced producer cells, and,subsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells. This media is then used to infect fibroblast cells.Media is removed from a subconfluent plate of fibroblasts and quicklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirtually all fibroblasts will be infected and no selection is required.If the titer is very low, then it may be necessary to use a retroviralvector that has a selectable marker, such as neo or his.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product.

Example 9 In vitro Angiogenesis Assay

This assay was used to determine the relative ability of TNF-gamma₁₂₋₁₄₇to inhibit the FGF-2-induced formation of capillary-like tubularstructures in cultures of adult bovine aortic endothelial (ABAE) cells.Three-dimensional collagen gel plates (24-well) were prepared byaddition of 0.5 ml chilled solution of 0.7 mg/ml of rat tail type Icollagen (Becton Dickinson Labwares, Bedford, Mass.) to each wellcontaining 1×DMEM and adjusting to neutral pH with NaHCO₃. Afterformation of collagen gel (about 1-2 mm thickness), ABAE cells wereseeded at 5×10⁴ cells/well. The cultures were maintained in a humidified5% CO₂ incubator at 37° C. in DMEM containing 10% calf serum (HyClone,Logan, Utah) supplemented with L-glutamine (2 mM) until the culturesreached confluence. The medium was then replaced with fresh mediumcontaining 20 ng/ml of FGF-2. The effect of TNF-gamma₁₂₋₁₄₇ as aninhibitor of FGF-2-induced formation of capillary-like tubularstructures in ABAE cultures was analyzed by supplementing the culturemedium with 0.1, 0.3, 1, 3, or 10 μg/ml of TNF-gamma₁₂₋₁₄₇. All cultureswere then maintained at 37° C. for an additional 48 hours and thendiscontinued by fixation with cold methanol (−20° C.).

The abundance of capillary-like structures formed by ABAE cells wasanalyzed by using a Kotron IBAS Image Analyzer assisted with a HamamatsuC2400 video camera and a Zeiss Axioshop microscope. The abundance of thecapillary-like structures were measured as percentages of the whiteareas over the total areas measured. As a control, the EC₅₀ value forthe angiogenic factor FGF-2 to stimulate in vitro angiogenesis was about5 ng/ml. As a further control, a maximum stimulatory effect was observedat 10 ng/ml of FGF-2.

As shown in FIG. 14 (in which TNF-gamma is designated “VEGI”),observable inhibition of FGF-2-induced tube formation in ABAE cultureswas observed by the addition of 1, 3, and 10 μg/ml of TNF-gamma₁₂₋₁₄₇(labeled as VEGI). The IC50 value for the inhibition of FGF-2-inducedtube formation was approximately 1 μg/ml, which was similar to thatobserved for the inhibition of endothelial cell growth (see Example 5).

Example 10 Chicken Embryonic Chorioallantoic Membrane (CAM) AngiogenesisAssay

The CAM assay was carried out essentially as described by Nguyen andcolleagues (Microvasc. Res. 47:31-40 (1994)) and Iruela-Arispe andDvorak (Thromb. Haemost. 78:672-677 (1997)). The method is based on thegrowth of new capillary vessels into a collagen gel pellet placeddirectly on the chorioallantoic membrane (CAM). The angiogenic factorsFGF-2 (100 ng) or VEGF (250 ng) were embedded in collagen gel pelletsand placed in contact with the CAM. Quantification of angiogenesis inthe gels was carried out 24 hours after the placement of the gel pelletsby using a Nikon fluorescence microscope. The images were transferred toa Power PC 100 AV, using a CCD Sony camera. Fluorescence intensity wasevaluated with NH Image 1.61 software. Fluorescence intensity for thepositive controls (which contained an angiogenic factor alone) wasconsidered as the maximum angiogenic response, and set, arbitrarily, at100. Due to the variability of the assay, inhibition greater than 20%was considered significant.

As an experimental determination of the effect of TNF-gamma on theFGF-2- or VEGF-induced angiogenesis, bacterially-produced TNF-gamma (250ng) was mixed with either FGF-2 (100 ng) or VEGF (250 ng) and embeddedin collagen gel pellets. The pellets were then placed in contact withthe CAM as described above. As shown in FIG. 15 (in which TNF-gamma isdesignated “VEGI”), TNF-gamma markedly inhibited new capillary growthinto collagen gels.

Example 11 In vivo Tumorigenicity Assay

An in vivo analysis of the potential effect of TNF-gamma on angiogenesiswas performed using a xenograft tumor model. In this experimentalapproach, one million human breast carcinoma cells (MDA-MB-231 orMDA-MB-435) were injected into the mammary fat pad of female nude miceeither alone or mixed with chinese hamster ovary (CHO) cells transfectedwith TNF-gamma or CHO cells transfected only with the CHO-vector (5×10⁶cells per mouse). The TNF-gamma polypeptide expressed in theseexperiments consisted of the polypeptide shown as SEQ ID NO:2 excludingthe N-terminal 22 amino acids. The N-terminal 22 amino acids of thisTNF-gamma mutein were replaced by the secretory signal peptide of humaninterleukin-6 (Hirano, T., et al., Nature 324:73-76 (1986)).

Mice which were coinjected with human breast carcinoma cells and eitherTNF-gamma-expressing CHO cells or vector-transfected CHO cells were thenrandomized and tumors were measured twice weekly. The tumor size wasassessed by measuring perpendicular diameters with a caliper andcalculated by multiplying the measurements of diameters in twodimensions. Data are presented in FIGS. 16A and 16B as the mean +/−standard deviation of six mice in each group.

Results presented in FIG. 16A and 16B (in which TNF-gamma is designated“VEGI”) illustrate the sizes of the MDA-MB-231 and MDA-MB-435,respectively, xenograft tumors (mm²) as a function of time (dayspostinnoculation). Tumors were measured beginning on day zero andapproximately at 5 day intervals through approximately the twenty-eighthday. In each case, tumors which resulted from breast carcinoma cellscoinjected with TNF-gamma-expressing CHO cells (represented by theclosed circles in FIGS. 16A and 16B) remained significantly smaller insize than those which resulted from breast carcinoma cells coinjectedwith vector-only CHO cells (represented by the open circles in FIGS. 16Aand 16B).

Example 12 Induction of NF-kB and c-Jun Kinase (JNK) by TNF-Gamma

Activation of cellular NF-kB is preceded by the phosphorylation,ubiquitination, and ultimate degradation of an endogenous NF-kBinhibitor molecule designated IkBa. Degradation of the inhibitor allowsthe p65 subunit of NF-kB to translocate to the nucleus where it can actas a transcriptional regulator. For this reason, a electrophoreticmobility shift analysis (EMSA) is an appropriate method for analyzingactivation of cellular NF-kB by treatment of cultured cells withTNF-gamma.

In these analyses cells (2×10⁶ per ml) were treated with differentconcentrations (0.1-1.0 μg/ml) of bacterially-produced TNF-gamma at 37°C. for 12 hours. Nuclear extracts were then prepared from the culturedcells and EMSA was performed as is well-known in the art and essentiallyas described (Singh, S. and Aggarwal, B. B. J. Biol. Chem.270:10631-10636 (1995)).

Treating U-937 cells with TNF-gamma for 12 hours resulted in increase inDNA-binding by the p65 subunit of NF-kB. Peak activation of DNA-bindingby p65 was observed when U-937 cells were treated with 1 μg/ml TNF-gammafor 12 hours. However, treatment of U-937 cells with as little as 0.2μg/ml TNF-gamma for 12 hours resulted in an observable increase in p65DNA-binding. TNF-gamma was observed to activate p65 DNA-binding overbasal levels from 30 minutes to 18 hours after the initiation oftreatment in U-937 cells.

These experiments were elaborated by determining a degradation profilefor IkBa in U-937 cells in response to treatment with TNF-gamma. A timecourse of IkBa degradation was determined by Western blot analysis, atechnique that is well-known by one of ordinary skill in the art and hasbeen described by Singh and Aggarwal (J. Biol. Chem. 270:24995-25000(1995)). IkBa was completely degraded when U-937 cells were treated with0.1 -1.0 μg/ml TNF-gamma for 12 hours.

The cellular kinase designated c-Jun kinase (JNK) is an early event incellular activation. The activation of JNK by TNF-gamma was analyzed asan additional method of determining cellular reaction to treatment withTNF-gamma. The JNK kinase activation assay is well-known by one of skillin the art and has been described by Derijard and colleagues (Cell76:1025-1029 (1994)). After treatment of U-937 cells with 0.1 to 3.0μg/ml of TNF-gamma for 12 hours, the cells were harvested and assayedfor JNK kinase activity. By 6 and 12 hours, JNK activity had increased2- and 3.6-fold, respectively.

Example 13 Effect of TNF-Gamma in Treating Adjuvant-induced Arthritis inRats

An analysis of the use of TNF-gamma to treat rheumatoid arthritis (RA)may be performed through the use of an adjuvant-induced arthritis (AIA)model in rats. AIA is a well-characterized and reproducible animal modelof rheumatoid arthritis which is well-known to one of ordinary skill inthe art (Pearson, Ann. Rheum. Dis. 15:379 (1956); Pearson & Wood,Arthritis Rheum. 2:440 (1959)). TNF-gamma is expected to inhibit theincrease in angiogensis or the increase in endothelial cellproliferation required to sustain the invading pannus in bone andcartilage observed in this animal model of RA. Lewis and BB rats(available from Charles River Lab, Raleigh, N.C. and the University ofMassachusetts Medical Center, Worcester, Mass.) are used as the commonand responsive strains for adjuvant-induced arthritis in theseexperiments.

Initiation of the arthritic condition is induced by the intradermalinjection of 0.1 ml adjuvant (5 mg/ml) into the base of the tail. Groupsof 5 to 6 rats receive either 0.1 to 1.0 mg/kg TNF-gamma or vehicleintra-articularly 20 days after the injection of adjuvant. At thistimepoint, acute inflammation reaches a maximal level and chronic pannusformation will have just begun. The effect of TNF-gamma on pannusformation is analyzed radiologically once each week after day 15following adjuvant challenge essentially as described by Taurog andcolleagues (J. Exp. Med. 162:962 (1985)). Briefly, rats are anesthetizedwith ether or chloral hydrate and positioned so that both hind limbs areX-rayed together. The X-ray films is examined blindly using a scoringsystem of 0-3 for periosteal reaction, bony erosions, joint spacenarrowing and destruction. When there is a significant amount of jointdamage in vehicle-treated rats, the animals are sacrificed. At thispoint, the paws are evaluated histologically for the relative degree oftissue damage and for the therapeutic effect TNF-gamma has elicited onthese joints.

Finally; TNF-gamma- and vehicle-treated animals undergo a clinicalevaluation twice per week to assess hind paw volume using aplethysmometer system and body weight.

Example 14 DR3 Ligand (TNF-Gamma) is a Novel Anti-tumor CytokineExisting in Two Different Forms and Differentially Expressed inDifferent Tissues and Cells

Background:

TNF (tumor necrosis factor) superfamily members play very importantroles in cell activation, proliferation, differentiation, apoptosis,cytotoxicity and immune regulation. Members of TNF ligand and receptorsuperfamily are often overexpressed in various human cancer cells and/oractivated lymphocytes, their extracellular accessibility makes themexcellent potential targets for specific antitumor therapy andimmunomodulating therapy. Over the past few years the list of moleculesbelonging to the TNF receptor and ligand superfamily has grown rapidly.The TNF ligand family of cytokines consist of over 13 type IItransmembrane proteins (except TNF-b), the TNF receptor superfamilyconsist of over 18 type I transmembrane proteins except OPG, also knownas OCIF or TR1, which is a secreted protein, and TRID/DcR1/TRIAL-R3,which is a GPI-linked cell surface molecule.

Several TNF receptor superfamily members as well as some of theintracellular signal transducers involved in apoptosis contain a stretchof amino acids, approximately 60 to 80 amino acid long, referred to asthe “death domain”. These death domain-containing receptors, such asTNFR1, Fas/Apo-1/CD95, DR3 (also known as Ws1, Apo3, TRAMP or LARD),DR4, DR5 or TRAIL-R2, upon activation by their ligands, recruit variousproteins that mediate cell death through the death domain. Theseproteins in turn recruit other proteins via their death domains or deatheffector domains to transduce the death signal. TNFR1 is expressed inmost tissues and cell types and is involved in transducing three majortypes of signals: activation of the transcription factor NF-kB, c-junN-terminal protein kinase and apoptosis. Whereas Fas is expressed inlymphocytes, liver, heart, lung, kidney, and ovary. In contrast, DR3 ispredominantly expressed in spleen, thymus, and peripheral bloodlymphocytes. The ligand for DR3 has not yet been identified. DR3interacts with TRADD, associates with RIP ordinarily only weakly, butassociates strongly when TRADD is overexpressed. In the presence ofTRADD, it also associates strongly with FADD. These results suggest thatthe mechanism of DR3-induced apoptosis is similar to that induced by Fasand TNFR1. Like TNFR1, DR3 also activates NF-kB.

We have identified several novel TNF receptor and ligand superfamilymembers using several search strategies. One novel TNF-like ligand,TNF-gamma, was predominantly expressed in endothelial cells. AlthoughTNF-gamma shares some of the activities TNF, it does not bind to TNFR1and TNFR2, indicating that TNF-gamma binds to a distinct receptor. Herewe show that TNF-gamma binds to DR3 in several receptor-ligand bindingassays. Interestingly, TNF-gamma exists in two different forms which aredifferentially expresses in different cells and tissues.

Results and Discussion:

We have identified several novel TNF receptor and ligand superfamilymembers from HGS database which contains over 1.5 million ESTs from over620 cDNA libraries. One novel TNF-like ligand predominantly expressed inan endothelial cell library exhibited 20-30% sequence homology to othermembers of the TNF family. The protein was named TNF-gamma-alpha (orVEGIa for Vascular Endothelial derived tumor Growth Inhibitor alpha).Subsequent database analysis and library screening identified a novelsplicing variant of TNF-gamma-alpha, designated TNF-gamma-beta (orVEGIb). This isoform was found predominantly in cDNA libraries of TNFa-and IL-1-induced endothelial cells, monocyte and activated T-cells. ThecDNA for TNF-gamma-alpha encodes 174 amino acid residues andTNF-gamma-beta encodes 251 amino acids. Both proteins havecharacteristics of type II transmembrane proteins. They only differ atthe N-terminus which corresponds to the intracellular and transmembranedomains (FIG. 18A-D and 19).

Recombinant TNF-gamma induces apoptosis in several cell lines such asbovine pulmonary artery endothelial cells and adult bovine aorticendothelial cells. [Bovine pulmonary artery endothelial cells wereincubated with various concentrations of TNF-gamma for 48 hours. Theapoptosis was assessed by nuclear staining with Hoechst 33342fluorescence dye (10 mg/ml).] TNF-gamma also induces nuclear factor kB(NF-kB) and c-Jun N-terminal kinase (JNK) activation, inhibitsangiogenesis in vitro. [U937 cells were transfected using lipofectamine(following manufacturers instruction) with 0.2 mg of reporter plasmid(NF-kB-SEAP). The transfected U937 cells were collected and added to the96-well plate (200 ml/well) with various concentrated of TNF-gamma.After Incubation at 37° C. for 72 hr, the NF-kB activity was measuredwith luminometer at absorbance of 450 nm.]

To identify the novel receptor and ligand pairs, several receptor-ligandbinding assays were established. Recombinant soluble TNF-gammacontaining the entire ectodomain binds to DR3-Fc fusion proteinimmobilized on BIAcore chip, purified DR3-Fc also binds to BIAcore chipimmobilized with TNF-gamma. [Purified DR3-Fc or TNF-gamma was analyzedon a BIAcore instrument flowcell derivatized with TNF-gamma or DR3-Fc.The shown data represents the net bound (off-rate) region of the plotafter binding of TNF-gamma to immobilized DR3-Fc receptor, or binding ofDR3-Fc to immobilized TNF-gamma, which is measured in relative massunits (RU) versus time. The binding conditions were performed at highreceptor chip densities under diffusion-limited conditions.] Usingimmunoprecipitation techniques, recombinant TNF-gamma wasco-immunoprecipated by DR3-Fc, but not LTbR-Fc immunoadhesins. [TheFc-extracellular domains of DR3 or Fc alone and the correspondingligands were prepared and binding assays were performed as describedelsewhere. The respective Fc-fusions were precipitated with proteinG-Sepharose and co-precipitated soluble ligands were detected byimmunoblotting with anti-TNF-gamma antibody. Bloting and detection wasperformed as described in BM Chemiluminescence Western Blotting kitprotocol.]

To further demonstrate the interaction between DR3 and TNF-gamma, wescreened several cell lines for cell surface expression of TNF-gammausing polyclonal antibody to recombinant soluble TNF-gamma. Consistentwith the Northern blot analysis, peripheral blood mononuclar cells(PBMC) and human umbilical vein endothelial cells (HUVEC) expressTNF-gamma on the cell surface by immunostaining with antibody toTNF-gamma. [Cells were collected by trypsinization or aspiration, andcentrifuged at 1500-2000 rpm for 5 min. The cell pellets wereresuspended and washed in 5 ml ice-cold PBS twice. The cells wereincubated for 30 min at 40° C. with antibody (10 mg/ml) to TNF-gamma todetected expression of TNF-gamma on cell surface, with DR3-Fc or LTbR-Fc(10 mg/ml) for receptor and ligand binding in the binding buffer (HBSScontaining 10% BSA, 20 mM HEPES, pH 7.2, 0.02% NaN3). Purified human IgG(25 mg/ml) was used as control. Cells were then washed and stained withphycoerythrin (PE) conjugated to goat anti-rabbit or anti-human IgG at20 mg/ml. Fluorescence was analyzed by a FACscan flow cytometer (BectonDickinson, Mountain View, Calif.).] Two tumor cell lines(MC-38/TNF-gamma and MDA-231/TNF-gamma) transfected with TNF-gamma alsoexpress TNF-gamma on the cell surface. FACS analysis showed that here isa shift in the most population following exposure MC-38/TNF-gamma cellsto DR3-Fc, indicating cell-surface binding between TNF-gamma and DR3.Similarly, a shift in the MDA-231 cells transfected with TNF-gamma wasobserved. In addition, DR3-Fc protein also binds to HUVEC cells andPBMC. It is noteworthy that DR3 expression and TNF-gamma binding to PBMCdeclined after prolonged stimulation with PHA. As predicated, DR3-Fcinhibits the TNF-gamma induced NF-kB activated in a dose-dependentmanner. [U937 cells were transfected using lipofectamine (followingmanafacturer's instructions) with 0.2 mg of reporter plasmid(NF-kB-SEAP). The transfected U937 cells were collected and added to the96-well plate (200 ml/well) with various concentration of DR3-Fcreceptor and 100 ng/ml of TNF-gamma. After incubation at 37° C. for 72hr. the NF-kB activity was measured with luminometer at absorbance of450 nm.]

TNF-gamma maps to the chromosomal location within band 9q32. Thischromosomal location is close to CD30L (9q33), but is different from thegenes for TNFα, LTα and LTβ which are tightly linked within the MHCcomplex on chromosome 6. Interestingly, the TNF-gamma receptor, DR3, wasassigned to the long arm of chromosome 1, region p36.2, is localized toa region where CD30, TNFR2 and OX40 have been mapped.

Consistent with the role of TNF-gamma and DR3 in apoptosis and immuneregulation as well as interaction of DR3 with TNF-gamma, localproduction of TNF-gamma caused complete tumor suppression in vivo in asyngeneic MC-38 murine colon cancer models. In the same animal model,local production of soluble DR3, which may block TNF-gamma function,promotes tumor growth. [The full-length TNF-gamma and extracellulardomain of DR3 was cloned into pcDNA3 expression vector and transfectedto MCA 38 cells, respectively. After selection and cloning, three clonesfrom each constructs were picked for tumorgenecity study. MCA 38 cells(1×10⁶ cells/mouse) expressing TNF-gamma or DR3 extracellular domainwere injected into C57BL6/6 mice. The tumor size was assessed bymeasuring perpendicular diameters with a caliper and calculated bymultipling the measurements of diameters in two dimensions. Data arerepresented as the mean +/− SD of 6 mice in each group.] It is clearthat most immune cells and cancer cells can express more than one TNFreceptor (even more than one death receptor) and ligand superfamilymember. The existence of multiple receptors for one ligand or multipleligands for one receptor, and multiple splicing variant forms ofreceptor or ligand suggests an unexpected complexity in the regulationof apoptosis and immune function. These receptors and ligands appear tobe functionally redundant, but their expression patterns are different,suggesting a distinct tissue or cell specific involvement in aparticular function. Moreover, the expression of these ligands andreceptors may differ at the level of individual cell types withintissues and the expression level on the same cell type may also differ.

It is estimated that 10% of genes can be alternatively spliced, but inmany cases the function of proteins produced remains obscure. To examinethe potential functional significance of the two splicing variants ofTNF-gamma, PCR analysis was performed in over 100 cDNA libraries. Theseresults are shown in the following table:

Differential Expression Pattern of DR3, TNF-Gamma-alpha, andTNF-Gamma-beta

Library DR3 TNF-ga TNF-gb Library DR3 TNF-ga TNF-gb Normal TissueAbnormal tissue and cell Liver + + Hepatocellular tumor + Lymph node + +Hodgkin's lymphoma + Tonsil + Rhabdomyosarcoma + Bone marrow + Nasalpolyps Spleen + Spleen, metastatic melanoma Heart + Spleen, chronicThymus + + lymphocytic leukemia Pericardium + Healing wound (skin) + +Brain + B-cell lymphoma Lung + Hemangiopericyloma Skeletal musclePancreas tumor + Placenta + Burned skin + Prostate + Prostrate cancer,stage C Pituitary U937 cell + Testis + + Ovarian tumor + Colon Coloncancer, metasticized + + Pancreas + to liver Kidney + Colon CancerKidney cortex + Crohn's disease Pualmonary Rejected kidney + +Adipose + + T-cell lymphoma + Ovary + + Ovary tumor CerebellumEndometrial tumor Hippocampus Skin tumor Hyperthalamus Pancreaticcarcinoma + Olfactory epithelium + + Jurkat cells + Striatumdepression + Hela cell line + + Pineal gland LNCAP + 0.3 nM androgen +LNCAP + 30 nM androgen + + Fetal tissue Normal cell 8 week embryo + +HUVEC + + + 9 week embryo + Dermal endothelial, + Fetal brain + + +Resting T cell Fetal kidney + + Activated T cell (12 hr) Fetalheart + + + Activated T cell (16 hr) + Fetal thymus + Activated T cell(24 hr) + + Fetal lung + + T cell helper I Fetal liver + T cell helperII + Fetal spleen + CD34+ + Primary dendritic cells, + EosinophilsMonocytes + + Osteoblasts Keratinocyte + + Stromal endometrial cellsStromal cell TF274

As shown in the table, DR3 and two forms of TNF-gamma are differentiallyexpressed in different tissues and cells. In the libraries tested, DR3was found to be expressed in most tissues, in activated T-cells,monocytes, dendritic cells, TH2 cells, and several other cell lines(such as U937, HeLa) and tumor tissues (such as hepatocellular tumor andHodgkin's lymphoma). DR3 expression was increased in LNCAP prostatecarcinoma cell line treated with 30 nM of synthetic androgen.TNF-gamma-alpha is only expressed in a few tissues or cells such asfetal brain, fetal heart, adipose, kidney cortex, olfactory epithelium,pancreatic carcinoma and HUVEC. In contrast, TNF-gamma-beta has a muchbroader expression pattern. At the cellular level, only endothelialcell, activated T-cells, monocytes, keratinocytes, HeLa and Jurkat cellsexpress TNF-gamma-beta. Only HUVEC, fetal brain, and fetal heart cDNAlibraries express both forms of TNF-gamma and DR3. TNF-gamma-alpha,TNF-gamma-beta, and DR3 are not expressed in resting T-cells or earlystage of activated T-cells (12 hr). DR3 becomes delectable at 16 hr, andboth DR3 and TNF-gamma-beta become detectable in T-cells at 24 hr afterPHA stimulation. The time-dependent induction of DR3 and thenTNF-gamma-beta in activated T-cells suggest that DR3 and TNF-gamma mayplay an import role in activation induced apoptosis.

Northern blot and cDNA database analysis indicated that DR3 expressionis found predominantly in tissues with high content of lymphocytes,TNF-gamma is predominantly expressed in endothelial cells, monocytes andactivated T-cells. Thus, DR3 and TNF-gamma may be involved in theactivation-induced apoptosis and the negative selection of lymphocytes.The expression pattern of DR3, TNF-gamma-alpha, and TNF-gamma-beta bydifferent cells and tissues. Expression of different splicing variantforms of DR3 or TNF-gamma is likely to set the balance betweensusceptibility and protection from DR3-mediated apoptosis. It is clearthat the pathway leading to apoptosis is highly regulated process andinvolving a series of proteins.

Another ligand for DR3, named as Apo3L has been described recently,which was also published as Tweak. Unlike TNF-gamma, Apo3L/Tweakexpressed in a wide variety of tissues. The interrelationship andfunctional importance between these two DR3 ligands remain to beinvestigated.

Conclusion:

One pair of novel receptor and ligand of TNF superfamily, DR3 andTNF-gamma, has been identified. Unlike other ligands of TNF family,TNF-gamma exists in two different forms and is differentially expressedin different cells and tissues. It has been suggested that one of themechanisms for regulating DR3 function is through alternative splicingof DR3. Alternative pre-mRNA splicing generates at least 11 isoforms ofDR3, providing a range of functional outcomes that may help shape theimmune response. Our data suggested that DR3 function can also beregulated through alternative splicing and differentially expression ofits ligand, TNF-gamma. These findings have great impact on how we viewthe regulation of apoptosis and TNF receptor superfamily function.Identification of two differentially expressed DR3 ligand variantsraised the possibility to selectively modulate apoptosis, immuneresponse and immune surveillance of tumor. Further characterization ofphysiological and pathological function of two differentially expressedTNF-gamma may provide new insights into the biological activities andphysiological function as well as therapeutical application of TNFreceptor and ligand superfamily. Understanding the role and mechanismsof action of these genes should allow us to develop ways to regulateapoptosis and cell proliferation in a variety of physiological andpathological conditions.

Materials and Methods:

Apoptosis Asassy:

Bovine pulmonary artery endothelia cells (BPAEC) were incubated withvarious concentrations of TNF-gamma for 48 hours. Apoptosis was assessedmorphologically and by nuclear staining with Hoechst 33342 fluorescencedye (10 mg/ml) in triplicate. Live and apoptotic cells were scored infour random fields, about 1,000 cells were counted. The DNAfragmentation was analysed as described previously.

BIAcore Receptor-ligand Binding Assay

Generation of recombinant receptor DR3-Fc fusion protein and recombinantTNF-gamma were described in previous papers. Purified TNF-gamma orDR3-Fc was immobilized on BIAcore respectively. Purified DR3-Fc orTNF-gamma was analyzed on a BIAcore instrument flowcell derivatized withTNF-gamma or DR3-Fc. The net bound (off-rate) region of the plot afterbinding of TNF-gamma to immobilized DR3-Fc receptor, or binding ofDR3-Fc to immobilized TNF-gamma, was measured in relative mass units(RU) versus time. The binding conditions were performed at high receptorchip densities under diffusion-limited conditions.

Co-Immunoprecipitation and Western Blot Analysis

Polyclonal antisera against TNF-gamma were prepared in rabbits asdescribed previously (Ni, J., et al., J. Biol. Chem. 272:10853-10858,(1997)). The Fc-extracellular domains of DR3 or Fc alone and thecorresponding ligands were prepared and binding assays were performed asdescribed elsewhere. The respective Fc-fusions were precipitated withprotein G-Sepharose and co-precipitated soluble ligands were detected byimmunoblotting with anti-TNF-gamma antibody. The samples were loadedinto a gel [NOVEX Pre-Cast Gels] (4˜20% Tris-Glycine Gel). Bloting anddetection was performed as described in BM Chemiluminescence WesternBlotting kit protocol.

FACS Analysis

Cells were collected by trypsinization or aspiration, and centrifuged at1500-2000 rpm for 5 min. The cell pellets were resuspended and washed in5 ml ice-cold PBS twice. The cells were incubated for 30 min at 40° C.with antibody (10 mg/ml) to TNF-gamma to detected expression ofTNF-gamma on cell surface, with DR3-Fc or LTbR-Fc (10 mg/ml) forreceptor and ligand binding in the binding buffer (HBSS containing 10%BSA, 20 mM HEPES, pH 7.2, 0.02% NaN3). Purified human IgG (25 mg/ml) wasused as a control. Cells were then washed and stained with phycoerythrin(PE) conjugated to goat anti-rabbit or anti-human IgG at 20 mg/ml.Fluorescence was analyzed by a FACscan flow cytometer (Becton Dickinson,Mountain View, Calif.).

NF-kB-SEAP (Secreted Alkaline Phospharase) Reporter Assay

U937 cells were transfected using lipofectamine (followingmanufacturer's instructions) with 0.2 mg of reporter plasmid(NF-kB-SEAP). The transfected U937 cells were collected and added to the96-well plate (200 ml/well) with various concentration of activeTNF-gamma or inactivated (boiled) TNF-gamma or in combination withvarious concentration DR3-Fc receptor and 100 ng/ml of TNF-gamma. AfterIncubation at 37° C. for 72 hr, the NF-kB activity was measured withluminometer at absorbance of 450 nm.

Tissue and Cell Distribution Analysis Using PCR on a Large Collection ofcDNA Libraries and cDNA Database:

To study the tissue distribution of DR3. TNF-gamma-alpha andTNF-gamma-beta, two gene specific primers were synthesized for eachgene. Over 100 cDNA libraries are tested and the libraries gave apositive predicted size signal are indicated as +.

In vivo Tumorigenecity Assay:

The full length TNF-gamma and extracellular domain of DR3 was clonedinto pcDNA3 expression vector (invitrogen, Carlsbad, Calif.) andtransfected to MCA 38 cells, respectively. Subsequent to transfection,G418 selection, and cloning, three clones from each constructs werepicked for tumorgenecity study. The expression of TNF-gamma and DR3 inMCA 38 cells were confirmed by Northern analysis. MCA 38 cells (1×10⁶cells/mouse) expressing TNF-gamma or DR3 extracellular domain wereinjected into C57BL6/6 mice. Mice then were randomiced and tumors weremeasured twice weekly. The tumor size was assessed by measuringperpendicular diameters with a caliper and calculated by multipling themeasurements of diameters in two dimensions. Data are represented as themean +/−SD of 6 mice in each group.

Example 15 TNF-Gamma-alpha, a Novel Member of TNF Cytokine Family,Causes Endothelial Cell Apoptosis

Background:

TNF-gamma-alpha is a novel protein with a molecular weight of 22 kD thatwas recently identified by searching the Human Genome Sciences (HGS)cDNA database (Tan,. K. B., et al, Gene 204:35-46 (1997)).TNF-gamma-alpha is a type II membrane protein and exhibits about 30%sequence homology to human tumor necrosis factor a (TNFa). This newlyidentified member of the TNF family has been demonstrated to beabundantly expressed in endothelial cells as well as in kidney, lung andprostate. TNF-gamma-alpha expression in HL-60 and THP1 cells was inducedby PMA treatment. Radiation hybrid mapping localized TNF-gamma gene onchromosome 9q32, near CD30L. Because of its overexpression inendothelial cells, TNF-gamma-alpha has been suggested to possibly play arole in vascular functions (Tan, K. B., et al, Gene 204:35-46). Thepresent study was undertaken to explore whether TNF-gamma-alpha inducesendothelial cell apoptosis, a phenomenon suggested to be one cause ofendothelial cell damage contributing to various inflammatory disordersand cardiovascular dysfunction (Bryant, D., et al, Circulation97:1375-1381. (1998)). To examine this possibility, we used bovinepulmonary artery endothelial cells (BPAEC) to which TNFa-inducedapoptosis has been demonstrated (Polunovsky, V. A., et al., Exp. CellRes. 214:584-594 (1994)). Apoptosis was detected on the basis ofmorphological (including ultrastrutual) and biochemical characteristics(DNA fragmentation). In addition, we studied the effects ofTNF-gamma-alpha on the activity of stress kinases, stress-activatedprotein kinase (SAPK/JNK) and p38 mitogen-activated protein kinase (p38MAPK), and the caspases. Both signaling pathways are believed to beimplicated in programmed cell death (Xia, Z., et al., Science270:1326-1331 (1995)). The expression of Fas and Bcl-2 inTNF-gamma-alpha-stimulated BPAEC was also determined in view of thedeath-promoting effect of Fas and the anti-apoptotic effect of Bcl-2(Nagata, S. and Golstein, P. Science 267:1449-1456 (1995)).

MATERIALS AND METHODS:

Materials.

TNF-gamma-alpha protein (22 kD) was provided by HGS. Ac-YVAD-AMC andAc-DEVD-AMC were purchased from American Peptide (Sunnyvale, Calif.,USA). ZVAD-fmk and Ac-YVAD-CHO were obtained from Enzyme Systems(Dublin, Calif., USA) and Peptides International (Louisville, Ky., USA),respectively. Ac-DQMD-AMC, Ac-LEED-AMC, Ac-VETD-AMC and anti-p38 MAPKmAb were provided by SmithKline Beecham (SB) Pharmaceuticals (King ofPrussia, Pa., USA). Ac-IETD-AMC and mouse-anti-human JNK mAb werepurchased from Biomol Research Laboratories (Plymouth Meeting, Pa., USA)and PharMingen (San Diego, Calif., USA), respectively. Mouse soluble TNFreceptor 1(sTNFR1) and TNF receptor 2 (sTNFR2) was obtained from R&DSystems (Minneapolis, Minn., USA).

Cell Cultures.

BPAEC were obtained from the American Type Culture Collection(Rockville, Md., USA). The cells were grown in DMEM supplemented with10% heat-inactivated FCS in a humidified environment of 5% CO₂/85% airat 37° C. as previously described (Yue, T. L., et al., Mol. Pharmacol.51:951-962 (1997)). Cells at a subconfluent density were used. Beforeexperiments, the medium was changed to DMEM contained 2% FCS. BPAEC frompassages 17-20 were used in all studies.

Morphological Assessment and Quantification of Apoptosis.

To quantify cells undergoing apoptosis, cell monolayers were fixed andstained with Hoechst 33324 (Molecular probe, Eugene, Oreg., USA) asdescribed previously (Yuc, T. L., et al., Mol. Pharmacol. 51:951-962(1997)). The morphological features of apoptosis (cell shrinkage,chromatin condensation, blebbing, and fragmentation) were monitored byfluorescence microscopy. Transmission electron microscopy study was doneas reported previously (Yue, T. L., et al., Mol. Pharmacol. 51:951-962(1997)).

DNA Fragmentation Analysis.

(a) DNA ladder: Cells treated with vehicle or TNF-gamma-alpha were lysedin lysis buffer containing 100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 2.5 mMEEDTA, 0.5% SDS, and 100 μg/ml protein kinase K. The lysates wereincubated at 55° C. for 16h. After incubation, the lysates were gentlyextracted three times with pheno/chloroform/soamyl alcohol, precipitatedin ethanol, treated with DNAse-free RNAse, re-extracted, andprecipitated again as described previously. DNA electrophoresis wascarried out in 1.8% agarose gels containing ethidium bromide, and DNAfragmentations were visualized under ultraviolet light.

(b) In situ end-labeling (TUNEL): BPAEC were cultured in two-chamberslides (Nunc) and treated with TNF-gamma-alpha for 8 to 24 h. In situdetection of apoptotic cells was performed by using terminaldeoxyribonucleotide transferase-mediated dUTP nick end labeling with anApopTag in situ apoptosis detection kit (Oncor) following themanufacturer's recommendation.

Stress-activated Protein Kinase (SAJPK/JNK) Assay.

SAPK activity was measured using GST-c-Jun₍₁₋₈₁₎ as bound toglutathione-Sepharose 4B as described previously (Yuc, T. L., et al.,Mol. Pharmacol. 51:951-962 (1997)). Briefly, the cells were treated withvehicle or TNF-gamma-alpha, washed, and lysed in lysis buffer. Thenuclear-free supernatant was normalized for protein content andimmunoprecipated with anti-SAPK antibody-conjugated Sepharose beads. Themixture was rotated 4° C. for 3 h. The phosphorylation buffer containingGST-c-Jun₍₁₋₈₁₎. 10 μC[g-³²P1-ATP, 125 μM ATP and 100 mM MgCl, was addedto the SAPK-bound beads in assay buffer. The reaction was terminatedafter 20 min at 30° C. by addition of protein loading buffer and heatedat 90° C. for 3 min. Phosphorylated proteins were resolved in 10%SDA-polyacrylamide gel electrophoresis followed by autoradiography. Theintensity of the bands was quantified by Phosphorimager (Yuc, T. L., etal., J. Mol. Cell. Cardiol. 30:495-507 (1998)).

p38 MAPK Assay.

The cell lysates prepared as above were immuno-precipitated wit anti-p38MAKP antibody bound to protein A agarose for 4 h at 4° C. The beads werewashed with lysis buffer and then with kinase buffer as describedpreviously (Kumar, S. M., et al., J. Biol. Chem. 271:30864-30869(1996)). The immune-complex kinase assay was initiated by the additionof 25 μl of kinase buffer containing 2 μg of GST-ATF2 and 50 μM [γ-³²P]ATP (20 Ci/mmol). The phosphorylated products were resolved by SDA-PAGEand visualized by Phosphorimage.

In vitro Transfection of Dominant-interfering Mutant of c-JUN in BPAEC.

The cells were plated in two-chamber slides. The cells werecotransfected with 0.5 μg/ml of Pegfp-c-1 (Clontech; Li, Y. and Horwitz,M. S. Biotechnology 23:1026-1028) as a fluorescent marker of transfectedcells together with 1 μg/ml of either the empty cloning vector pcDNA1(control) or the dominant-interfering c-Jun mutant pcDNA1-FigΔ169 (Xia,Z., et al., Science 270:1326-1331 (1995)) using Calphos MaximizerTransfection Kit (Clontech) according to the manufacturer'srecommendation. Following transfection, the cells were allowed torecover in complete medium for 24 h. The cells were treated withTNF-gamma-alpha and the number of apoptotic cells was assessed bynuclear staining after fixation as described in Methods.

Caspase Activity Assay.

The cells were treated with vehicle of TNF-gamma-alpha. Caspase activityassays were performed as reported previously (Yuc, T. L., et al.,supra). Briefly, cells were harvested and suspended in buffer containing25 mM HEPES, pH 7.5, 10% sucrose, 0.1% CHAPS, 2 mM DDT, 5 mM PMSF, and 1μM pepstatin A. The suspension was forced through a 25 gauge needle 10times to break cells the homogenate was centrifuged at 100,000×g for 1h, and the cleared lysates were used for enzyme assays. Cell extracts(5-20 μg protein) were diluted into the assay buffer (Table 2) andpreincubated for 10 min at 30° C. prior to the addition of thesubstrates. Levels of released 7-amino-4-methylcocmarin (AMC) weremeasured with a Cytofluor-4000 fluorescent plate reader (PerseptiveBiosystems) at an excitation and emission wavelengths of 360 nm and 460nm, respectively.

Immunohistochemical Analysis for Fas, Bcl-2 and Caspase-3 Expression.

The cells were cultured in two-chamber slides. After treatment withvehicle or TNF-gamma-alpha, the cells were fixed with 4%paraformaldehyde for 30 min at 4° C. and then changed to cold PBS. Thecells were treated with 0.2% Triton X-100 for 40 min at 4° C., washedwith cold PBS and then non-specific immunoglobulin binding sites wereblocked with normal goat serum (Vector Laboratories) for 1 h at roomtemperature. The cell samples were incubated with the primary antibodymouse anti-human Fas (Upstate Biotechnology), mouse anti-human Bcl-2(DAKO) or rabbit anti-human CPP32 p17 peptide polyclonal antisera(SmithKline Beecham), for 1 h at room temperature. As a negativecontrol, the cell samples were incubated with nonimmune IgG (for Bcl-2and CPP32) or IgM (for Fas) instead of the primary antibody. Afterincubation with the primary antibody, cells were washed with PBS andthen incubated for 30 min with a secondary antibody conjugated tofluorscein isothiocyanate. Cells were washed, treated with Veetashieldmounting medium (Vector Laboratories) and viewed by fluorescencemicroscopy (Olympus IX70).

Statistical Analysis.

All values are represented as mean ±S.E.M. of n independent experiments.Statistical evaluation was performed by using one-way analysis ofvariance. Differences with a value of p<0.05 were consideredsignificant.

Results:

TNF-gamma-alpha Induces Apoptosis in BPAEC.

When BPAEC were exposed to TNF-gamma-alpha the cells shrunk andretracted from their neighboring cells, and the cytoplasma becamecondensed. Cells stained with Hoechst 33324 and assessed by fluorescencemicroscopy demonstrated condensed chromatin of fragmented nuclei andblebbing of the plasma membrane. The study with transmission electronmicroscopy showed that TNF-gamma-alpha-treated BPAEC contained manycells undergoing morphologic alterations characteristic of apoptosisincluding condensation of chromatin and appearance of apoptotic bodies.The characteristic degradation of DNA into oligonucleosomal-lengthfragmentation was observed when the cells were exposed toTNF-gamma-alpha (30-300 ng/ml) for 24 h. The DNA fragments in situ wasfurther visualized by using TUNEL method. A considerable fraction ofendothelial cells treated with TNF-gamma-alpha showed positive staining;no positively stained cells were found in the vehicle-treated cultures.

TNF-gamma-alpha-induced endothelial cell apoptosis was a time- andconcentration-dependent process with an EC₃₀ value of 72 ng/ml. Asignificant increase in the number of cells with apoptotic morphologicalchanges was apparent 6-8 h after exposure of the cells toTNF-gamma-alpha. Under similar conditions, TNF-a at 10 ng/ml inducedapoptosis in PEAPC by 16.7±3.2% (n=4).

Effects of sTNFR1 and sTNFR2 on TNF-Gamma-alpha-induced Apoptosis inBPAEC.

Neither sTNFR1 nor sTNFR2 showed effect on TNF-gamma-alpha-inducedapoptosis in BPAEC. Under the same condition TNFa-induced apoptosis inBPAHC was reduced by sTNFR1 significantly.

Regulation of Fas and Bcl-2 Expression in Endothelial Cells byTNF-gamma-alpha. Immunocytochemical analysis of Fas and Bcl-2 proteinswas determined at 8 and 24 h after treatment with TNF-gamma-alpha. Thebasal level of Fas in BPAEC was undetectable. However, a significantnumber of cells expressing Fas receptor were detected at 8 and 24 hafter stimulation. When mouse IgM was substituted for the primaryantibody, positive Fas immunoreactivity was not detected. In contrast,Bcl-2 expression was not detected in neither unstimulated norTNF-gamma-alpha-treated BPAEC.

Activation of SAPK/JNK and p-38 MAPK.

With regard to the effects of TNF-gamma-alpha on SAPK/JNK activity inBPAEC, exposure of endothelial cells to TNF-gamma-alpha induced a rapidactivation of SAPK/JNK. A significant increase in SAPK/JNK activity wasdetected 20 min after stimulation, peaked at 40 min. and then returnedto the basal levels after 60 min. TNF-gamma-alpha-induced activation ofSAPK/JNK in endothelial cells is a concentration-dependent process. Somebasal activities of SAPK/JNK activity was increased by 5.6±1.4 folds(p<0.05 n=4) and 9.1±1.8 folds (p<0.01 n=6) over basal level in thepresence of 50 and 300 ng/ml of TNF-gamma-alpha, respectively.TNF-gamma-alpha activated p38 MAPK in BPAEC with a similar time courseas SAPK/JNK but to a lesser extent. The peak of p38 MAKP activity wasincreased by 3.1±0.5 and 3.8±0.4 folds over the basal level in thepresence of 100 and 300 ng/ml of TNF-gamma-alpha, respectively.

Effects on TNF-Gamma-alpha-induced Apoptosis by Expression ofDominant-interfering Mutant of c-JUN in BPAEC or by the p38 MAPKInhibitor, SB203580.

To investigate the role of SAPK/JNK in TNF-gamma-alpha-induced apoptosisin BPAEC, we transfected BPAEC with a dominant-interfering mutant ofc-JUN, pcDNA1-FlagΔ169, in which a deletion in the NH2-terminaltransactivation domain that includes the binding site for JNK (Xia, Z.,et al., supra). Expression of dominant-interfering c-JUN construct inBPAEC reduced TNF-gamma-alpha-induced apoptosis by 62.8% (p<0.05).TNF-gamma-alpha-induced apoptosis in BPAEC was also attenuated by aspecific p38 MAPK inhibitor, SB203580, in a concentration dependentmanner. In the presence of 3 and 10 μM of SB203580,TNF-gamma-alpha-induced BPAEC apoptosis was reduced by 33% (p<0.05) and51% (p<0.01), respectively. No further inhibition was observed when theconcentration of SB203580 was increased.

Activation of Caspases in BPAEC by TNF-Gamma-alpha.

TNF-gamma-alpha-induced BPAEC apoptosis was attenuated by ZVAD-fmk, anirreversible cell-permeable inhibitor of caspase (Jocobson, N. L., etat., Cell Biol. 133:1041-1051 (1996)), added to the culture medium 1 hprior to TNF-gamma-alpha treatment. Under the same conditions, theaddition of Ac-YYAD-CHO, a relatively specific inhibitor of caspase-1(Thorberry, N. A., et al., Nature (Lond) 356:768-774 (1992)), up to 100μM showed no effect in enhancing BPAEC rescue. To further determinewhich of the caspase family members are activated in theTNF-gamma-alpha-induced apoptotic process in the endothelial cells, weexamined cell extracts for protcolytic activity. The relative rates ofAMC formation were measured with a series of defined peptide sequencevariants that are relatively specific for caspase 1, 3, 4, 7, or 8 underthe optimal conditions as described previously (Yuc, T. L., et al.,supra). Similar results were observed from three repeated experiments.Cell extracts from TNF-gamma-alpha-treated BPAEC were highly active onAc-DEVD-AMC and to a lesser extent on Ac-DQMD-AMC, but not active on theremaining three substrates which are more specific for caspase 1, 4, and8. The proteolytic activity appeared at 6 h after the cells were treatedwith TNF-gamma-alpha, peaked at 24 h, and gradually returned to basallevels within 48 h. The relative velocities of four substrate hydrolysisrates by the TNF-gamma-alpha-treated cell extracts and recombinantcaspase-3 were compared. The relative velocities of the two enzymesources of four substrates were very similar.

To further confirm that caspase-3 is activated by TNF-gamma-alpha inBPAEC, immunocytochemical detection of its enzymatically active form,the 17-kD subunit, was performed. The antibody was raised against apeptide from the C-terminal portion of the p17 subunit. The neoepitopeantibody only binds caspase-3 if there has been specific cleavagebetween the “p-10” and “p-20” subunits. Using this neoepitope antibody,only processed caspase-3 is detected, but not the porenzyme (Yuc, T. L.,et al., supra). The 17 kD subunit of caspase-3 was detected inTNF-gamma-alpha-treated but not vehicle-treated BPAEC, and was localizedwith fragmented nuclei within the cells.

Discussion:

The studies presented in this paper demonstrate that TNF-gamma-alpha, anovel TNF-like cytokine and a type II transmembrane protein, inducesintensive apoptosis in cultured endothelial cells as reflected bymorphological and biochemical criteria. Under our experimentalconditions, spontaneous BPAEC death was approximately 2-4% which is inaccord with a previous observation (Polunovsky, V. A., et al., supra).The effect of TNF-gamma-alpha was concentration-dependent with an EC₈₀value of 72 ng/ml (3.5 nM) and a significant number of apoptotic cellswas detected 6-8 h after treatment. Moreover, the expression ofproapoptotic gene, Fas, was demonstrated in TNF-gamma-alpha-treatedBPAEC, which is consistent with that observed in apoptotic endothelialcells reported previously (Yuc, T. L., et al., supra).

The receptor(s) mediating TNF-gamma-alpha activity has not beenidentified as yet. To examine whether TNF-gamma-alpha acts via distinctreceptor(s), we tested the effects of sTNFR1 and sTNFR2 onTNF-gamma-alpha-induced apoptosis in BPAEC. These two TNFRs have beenshown previously to block the cell surface TNFR1 and TNFR2 mediated TNFbioactivities on responsive cell lines (data from R&D Systems). NeithersTNFR1 nor sTNFR2 inhibited the effect of TNF-gamma-alpha on BPAEC. Incontrast, TNFa-induced apoptosis in BPAEC was significantly reduced bysTNF1. The results suggest clearly that TNF-gamma-alpha-induced celldeath is independent of sTNFR1 or TNFR2.

Recent research efforts on TNF family members have demonstrated thatTNFa and Fas activate stress protein kinases, SAPK/JNK and p38 MAPK, ina variety of cell types (Sluss, H. K., et al., Cell Biol. 14:8376-8384(1994)), however, the effects of other members of this family on SAPKand p38 MAPK are not well studies. Moreover, controversies regarding therole of SAPK/JNK and p38 MAPK in TNFa or Fas-mediated cell death havebeen reported. For example, TNFa-induced apoptosis is dependent on JNKactivity in U937 cells (Verjeij, M., et al., Nature (Lond) 380:75-79(1995); Zanke, B. W., et al., Curr. Biol. 6:606-613 (1996)) but not infibroblasts (Reinhard, C., et al., EMBO J. 16:1080-1092 (1997))indicating that the consequences of JNK activation vary considerablyamong cell types. Fas-mediated JNK activation occurs with a differentkinetics from that of TNFa, suggesting that TNFa and Fas most likelyactivate JNK through a different mechanism (Wilson, D. J., et al., Eur.J. Immunol. 26:989-994 (1996)). Moreover, Juo, et al., reported recentlythat blockade of p38 MAPK by a specific p38 MAPK inhibitor did notaffect Fas-mediated .apoptosis in Jurkat cells (Juo, P., et al., Mol.Cell Biol. 17:24-35 (1997)). Therefore, we were interested in findingwhether TNF-gamma-alpha activates JNK and p38 MAPK, and what is the roleof this activation in TNF-gamma-alpha-mediated apoptosis in BPAEC. Thepresent investigation clearly demonstrates that both JNK and p38 MAPKwere rapidly activated by TNF-gamma-alpha in a similar fashion asobserved in TNFa-activated U937. Moreover, expression ofdominant-interfering mutant of c-JUN in BPAEC reducedTNF-gamma-alpha-induced cell death indicating thatTNF-gamma-alpha-induced apoptosis in BPAEC was dependent on JNKactivity. To address the potential involvement of p38 MAPK inTNF-gamma-alpha-mediated apoptosis in BPAEC, a specific p38 MAPKinhibitor SB203580 was tested. This inhibitor has been shown tospecifically inhibit p38 MAPK activity in vitro with no effect on avariety of kinases tested, including JNK and ERK-1 (Cuenda, A., et al.,FEBS Lett. 364:229-233 (1995)). TNF-gamma-alpha-induced apoptosis inBPAEC was also reduced by SB203580 in a concentration-dependent manner,indicating that p38 MAPK signaling pathway is involved inTNF-gamma-alpha-mediated BPAEC apoptosis. This effect is different fromthat observed in Fas-mediated apoptosis in Jurkat cells in whichSB203580 had no protective effect (Juo, P., et al., supra). Moreover,TNF-gamma-alpha-induced p38 MAPK activation occurs with must fasterkinetics in BPAEC than that observed in Jurkat cells in which the peakof p38 MAPK activation was at 24 h after stimulation by Fas, indicatingTNF-gamma-alpha and Fas most likely activate p38 MAPK through adifferent mechanism with a different outcome. Our data further suggeststhat different members of TNF family may have different signalingpathways to mediate cell death or have different effects in differentcell types.

Recent work has supported a central role for the caspase family members,as effectors of apoptosis (Kumar, S. M., et al., supra). However, therole of caspases in endothelial cell apoptosis has not been sufficientlyexplored. Two characteristic features of the caspase family have beenelucidated; they cleave their target proteins after specific asparticacids, resulting in two subunits that together form the active site ofthe enzyme (Nicholson, D. W., et al., Nature (Lond) 376:3743 (1995);Kumar, S. M., et al., supra). Among the caspase family, caspase-3(CPP32) has been considered as a central component of the proteolyticcascade during apoptosis and plays a key role in this family (Wang, X.,EMBO J. 15: 1012-1020 (1996); Woo, M., et al., Gene Development12:806-819 (1998)). TNF-gamma-alpha-induced BPAEC apoptosis wasinhibited by ZVAD-fmk, indicating a potential role for the caspasefamily in this effector pathway for apoptosis. To determine which of thecaspase family members are involved, we examined the substratespecificity of proteolytic activity in the extracts fromTNF-gamma-alpha-activated BPAEC by measuring the relative rate of AMCformation from 6 different substrates which are relatively specific forcaspases 1, 3, 4, 7 and 8 (Talanian, R. V., et al., J. Biol. Chem.272:9677-9682 (1997)). Treatment of BPAEC with TNF-gamma-alpha resultedin a significant increase in protcolytic activity towards DEVD-AMCmainly and DQMD-AMC to some extent, both of which show the relativespecificity for caspase-3 (Kumar, S. M., et al., supra). There was noinduction in protcolytic activity in TNF-gamma-alpha-activated cellextracts when Ac-YVAD-AMC, LEED-AMC or VETD-AMC were used as thesubstrate, indicating that caspases 1, 4 and 8 might not be involved.Moreover, comparison of the substrate specificity of the extracts fromTNF-gamma-alpha-treated BPAEC with recombinant caspase-3 showed asimilar pattern, further suggesting that caspase-3 may be thepredominant member in the caspase family activated by TNF-gamma-alpha.Furthermore, immunocytochemical studies detected the active form ofcaspase-3 in TNF-gamma-alpha treated BPAEC. It was reported thatmultiple caspase homologues were found in both the cytoplasm and nucleusin etoposid-induced apoptosis in HL-60 cells (Martins, I. M., et al., J.Biol. Chem. 272:7421-7430 (1997)). Interestingly, inTNF-gamma-alpha-induced apoptotic BPAEC the immunoreactive 17kD subunitof caspase-3 was only localized with fragmented nuclei, furtherindicating a role of caspase-3 in TNF-gamma-alpha-induced apoptosis.Whether this active caspase-3 was transported into the nucleus or theinactive caspase-3 is already in the nucleus awaiting activationpromoted by TNF-gamma-alpha requires further investigation. Takentogether, these results suggest that caspase-3 was activated byTNF-gamma-alpha-induced cell apoptosis. However, our results cannotexclude other members of this family, especially those closely relatedto caspase-3, such as caspase-7, in mediating TNF-gamma-alpha-inducedapoptosis. Moreover, ZVA-fmk was less effective at the later time (30 h)compared to the earlier time (14 h) for inhibitingTNF-gamma-alpha-included apoptosis in BPAEC, suggesting acaspase-independent of negative-feedback mechanism may exist at thelater phase of TNF-gamma-alpha-induced BPAEC apoptosis.

In summary, the present studies have demonstrated that TNF-gamma-alpha,a novel member of TNF cytokine family, causes endothelial cellapoptosis. TNF-gamma-alpha appears to act through a receptor which isdistinct from TNF receptors 1 or 2. The effect of TNF-gamma-alpha is viaactivation of the stress protein kinases, SAPK/JNK and p38 MAPK, and thecaspases, mainly caspase-3 like protease. Apoptotic programmed celldeath has been suggested to be a cause of endothelial cell damagecontributing to various inflammatory disorders and cardiovascular injury(Karsan, A. Trends Cardiovasc. Med. 8:19-24 (1998)). Moreover,endothelial cell apoptosis may be an important mechanism involved in abalance between antiangiogenic and proangiogenic processes, and loss ofthis balance will lead to a variety of diseases such as solid tumormetastasis and retinopathy (Folkman, J. and Shing, J. J. Biol. Chem.267:10931-10934 (1992); Brooks, P. C., et al., Cell 79:1157-1164(1994)).

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

The entire disclosure of all publications (including patents, patentapplications, journal articles, laboratory manuals, books, or otherdocuments) cited herein are hereby incorporated by reference.

Further, the Sequence Listing submitted herewith in both computer andpaper forms are hereby incorporated by reference in their entireties.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 24 <210> SEQ ID NO 1 <211>LENGTH: 2442 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (783)..(1304) <221> NAME/KEY:mat_peptide <222> LOCATION: (864)..(1304) <221> NAME/KEY: sig_peptide<222> LOCATION: (783)..(863) <221> NAME/KEY: misc_feature <222>LOCATION: (2265) <223> OTHER INFORMATION: n equals a, t, g, or c <221>NAME/KEY: misc_feature <222> LOCATION: (2273) <223> OTHER INFORMATION: nequals a, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (2307)<223> OTHER INFORMATION: n equals a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (2336) <223> OTHER INFORMATION: n equals a,t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (2341) <223>OTHER INFORMATION: n equals a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (2379) <223> OTHER INFORMATION: n equals a, t, g, or c<400> SEQUENCE: 1 cccaatcaag agaaattcca tactatcacc agttggccga ctttccaagtctagtgcaga 60 aatccaaggc 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tccttg 1211 Gly Ser Asn Trp Phe Gln Pro Ile Tyr Leu Gly Ala Met Phe Ser Leu105 110 115 caa gaa ggg gac aag cta atg gtg aac gtc agt gac atc tct ttggtg 1259 Gln Glu Gly Asp Lys Leu Met Val Asn Val Ser Asp Ile Ser Leu Val120 125 130 gat tac aca aaa gaa gat aaa acc ttc ttt gga gcc ttc tta cta1304 Asp Tyr Thr Lys Glu Asp Lys Thr Phe Phe Gly Ala Phe Leu Leu 135 140145 taggaggaga gcaaatatca ttatatgaaa gtcctctgcc accgagttcc taattttctt1364 tgttcaaatg taattataac caggggtttt cttggggccg ggagtagggg gcattccaca1424 gggacaacgg tttagctatg aaatttgggg ccaaaatttc acacttcatg tgccttactg1484 atgagagtac taactggaaa aaggctgaag agagcaaata tattattaag atgggttgga1544 ggattggcga gtttctaaat attaagacac tgatcactaa atgaatggat gatctactcg1604 ggtcaggatt gaaagagaaa tatttcaaca cctccctgct atacaatggt caccagtggt1664 ccagttattg ttcaatttga tcataaattt gcttcaattc aggagctttg aaggaagtcc1724 aaggaaagct ctagaaaaca gtataaactt tcagaggcaa aatccttcac caatttttcc1784 acatactttc atgccttgcc taaaaaaaat gaaaagagag ttggtatgtc tcatgaatgt1844 tcacacagaa ggagttggtt ttcatgtcat ctacagcata tgagaaaagc tacctttctt1904 ttgattatgt acacagatat ctaaataagg aagtttgagt ttcacatgta tatcccaaat1964 acaacagttg cttgtattca gtagagtttt cttgcccacc tattttgtgc tgggttctac2024 cttaacccag aagacactat gaaaaacaag acagactcca ctcaaaattt atatgaacac2084 cactagatac ttcctgatca aacatcagtc aacatactct aaagaataac tccaagtctt2144 ggccaggcgc agtggctcac acctgtaatc ccaacacttt gggaggccaa ggtgggtgga2204 tcatctaagg ccgggagttc aagaccagcc tgaccaacgt ggagaaaccc catctctact2264 naaaatacna aattagccgg gcgtggtagc gcatggctgt aancctggct actcaggagg2324 ccgaggcaga anaattnctt gaactgggga ggcagaggtt gcggtgagcc cagancgcgc2384 cattgcactc cagcctgggt aacaagagca aaactctgtc caaaaaaaaa aaaaaaaa2442 <210> SEQ ID NO 2 <211> LENGTH: 174 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <400> SEQUENCE: 2 Met Arg Arg Phe Leu Ser Lys Val Tyr SerPhe Pro Met Arg Lys Leu -25 -20 -15 Ile Leu Phe Leu Val Phe Pro Val ValArg Gln Thr Pro Thr Gln His -10 -5 -1 1 5 Phe Lys Asn Gln Phe Pro AlaLeu His Trp Glu His Glu Leu Gly Leu 10 15 20 Ala Phe Thr Lys Asn Arg MetAsn Tyr Thr Asn Lys Phe Leu Leu Ile 25 30 35 Pro Glu Ser Gly Asp Tyr PheIle Tyr Ser Gln Val Thr Phe Arg Gly 40 45 50 Met Thr Ser Glu Cys Ser GluIle Arg Gln Ala Gly Arg Pro Asn Lys 55 60 65 Pro Asp Ser Ile Thr Val ValIle Thr Lys Val Thr Asp Ser Tyr Pro 70 75 80 85 Glu Pro Thr Gln Leu LeuMet Gly Thr Lys Ser Val Cys Glu Val Gly 90 95 100 Ser Asn Trp Phe GlnPro Ile Tyr Leu Gly Ala Met Phe Ser Leu Gln 105 110 115 Glu Gly Asp LysLeu Met Val Asn Val Ser Asp Ile Ser Leu Val Asp 120 125 130 Tyr Thr LysGlu Asp Lys Thr Phe Phe Gly Ala Phe Leu Leu 135 140 145 <210> SEQ ID NO3 <211> LENGTH: 233 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400>SEQUENCE: 3 Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu GluAla 1 5 10 15 Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg CysLeu Phe 20 25 30 Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr ThrLeu Phe 35 40 45 Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu GluSer Pro 50 55 60 Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln Ala Val ArgSer Ser 65 70 75 80 Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val ValAla Asn Pro 85 90 95 Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg AlaAsn Ala Leu 100 105 110 Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln LeuVal Val Pro Ser 115 120 125 Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val LeuPhe Lys Gly Gln Gly 130 135 140 Cys Pro Ser Thr His Val Leu Leu Thr HisThr Ile Ser Arg Ile Ala 145 150 155 160 Val Ser Tyr Gln Thr Lys Val AsnLeu Leu Ser Ala Ile Lys Ser Pro 165 170 175 Cys Gln Arg Glu Thr Pro GluGly Ala Glu Ala Lys Pro Trp Tyr Glu 180 185 190 Pro Ile Tyr Leu Gly GlyVal Phe Gln Leu Glu Lys Gly Asp Arg Leu 195 200 205 Ser Ala Glu Ile AsnArg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly 210 215 220 Gln Val Tyr PheGly Ile Ile Ala Leu 225 230 <210> SEQ ID NO 4 <211> LENGTH: 205 <212>TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 4 Met Thr Pro ProGlu Arg Leu Phe Leu Pro Arg Val Cys Gly Thr Thr 1 5 10 15 Leu His LeuLeu Leu Leu Gly Leu Leu Leu Val Leu Leu Pro Gly Ala 20 25 30 Gln Gly LeuPro Gly Val Gly Leu Thr Pro Ser Ala Ala Gln Thr Ala 35 40 45 Arg Gln HisPro Lys Met His Leu Ala His Ser Thr Leu Lys Pro Ala 50 55 60 Ala His LeuIle Gly Asp Pro Ser Lys Gln Asn Ser Leu Leu Trp Arg 65 70 75 80 Ala AsnThr Asp Arg Ala Phe Leu Gln Asp Gly Phe Ser Leu Ser Asn 85 90 95 Asn SerLeu Leu Val Pro Thr Ser Gly Ile Tyr Phe Val Tyr Ser Gln 100 105 110 ValVal Phe Ser Gly Lys Ala Tyr Ser Pro Lys Ala Pro Ser Ser Pro 115 120 125Leu Tyr Leu Ala His Glu Val Gln Leu Phe Ser Ser Gln Tyr Pro Phe 130 135140 His Val Pro Leu Leu Ser Ser Gln Lys Met Val Tyr Pro Gly Leu Gln 145150 155 160 Glu Pro Trp Leu His Ser Met Tyr His Gly Ala Ala Phe Gln LeuThr 165 170 175 Gln Gly Asp Gln Leu Ser Thr His Thr Asp Gly Ile Pro HisLeu Val 180 185 190 Leu Ser Pro Ser Thr Val Phe Phe Gly Ala Phe Ala Leu195 200 205 <210> SEQ ID NO 5 <211> LENGTH: 244 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <400> SEQUENCE: 5 Met Gly Ala Leu Gly Leu Glu GlyArg Gly Gly Arg Leu Gln Gly Arg 1 5 10 15 Gly Ser Leu Leu Leu Ala ValAla Gly Ala Thr Ser Leu Val Thr Leu 20 25 30 Leu Leu Ala Val Pro Ile ThrVal Leu Ala Val Leu Ala Leu Val Pro 35 40 45 Gln Asp Gln Gly Gly Leu ValThr Glu Thr Ala Asp Pro Gly Ala Gln 50 55 60 Ala Gln Gln Gly Leu Gly PheGln Lys Leu Pro Glu Glu Glu Pro Glu 65 70 75 80 Thr Asp Leu Ser Pro GlyLeu Pro Ala Ala His Leu Ile Gly Ala Pro 85 90 95 Leu Lys Gly Gln Gly LeuGly Trp Glu Thr Thr Lys Glu Gln Ala Phe 100 105 110 Leu Thr Ser Gly ThrGln Phe Ser Asp Ala Glu Gly Leu Ala Leu Pro 115 120 125 Gln Asp Gly LeuTyr Tyr Leu Tyr Cys Leu Val Gly Tyr Arg Gly Arg 130 135 140 Ala Pro ProGly Gly Gly Asp Pro Gln Gly Arg Ser Val Thr Leu Arg 145 150 155 160 SerSer Leu Tyr Arg Ala Gly Gly Ala Tyr Gly Pro Gly Thr Pro Glu 165 170 175Leu Leu Leu Glu Gly Ala Glu Thr Val Thr Pro Val Leu Asp Pro Ala 180 185190 Arg Arg Gln Gly Tyr Gly Pro Leu Trp Tyr Thr Ser Val Gly Phe Gly 195200 205 Gly Leu Val Gln Leu Arg Arg Gly Glu Arg Val Tyr Val Asn Ile Ser210 215 220 His Pro Asp Met Val Asp Phe Ala Arg Gly Lys Thr Phe Phe GlyAla 225 230 235 240 Val Met Val Gly <210> SEQ ID NO 6 <211> LENGTH: 278<212> TYPE: PRT <213> ORGANISM: Rattus norvegicus <400> SEQUENCE: 6 MetGln Gln Pro Val Asn Tyr Pro Cys Pro Gln Ile Tyr Trp Val Asp 1 5 10 15Ser Ser Ala Thr Ser Pro Trp Ala Pro Pro Gly Ser Val Phe Ser Cys 20 25 30Pro Ser Ser Gly Pro Arg Gly Pro Gly Gln Arg Arg Pro Pro Pro Pro 35 40 45Pro Pro Pro Pro Ser Pro Leu Pro Pro Pro Ser Gln Pro Pro Pro Leu 50 55 60Pro Pro Leu Ser Pro Leu Lys Lys Lys Asp Asn Ile Glu Leu Trp Leu 65 70 7580 Pro Val Ile Phe Phe Met Val Leu Val Ala Leu Val Gly Met Gly Leu 85 9095 Gly Met Tyr Gln Leu Phe His Leu Gln Lys Glu Leu Ala Glu Leu Arg 100105 110 Glu Phe Thr Asn His Ser Leu Arg Val Ser Ser Phe Glu Lys Gln Ile115 120 125 Ala Asn Pro Ser Thr Pro Ser Glu Thr Lys Lys Pro Arg Ser ValAla 130 135 140 His Leu Thr Gly Asn Pro Arg Ser Arg Ser Ile Pro Leu GluTrp Glu 145 150 155 160 Asp Thr Tyr Gly Thr Ala Leu Ile Ser Gly Val LysTyr Lys Lys Gly 165 170 175 Gly Leu Val Ile Asn Glu Ala Gly Leu Tyr PheVal Tyr Ser Lys Val 180 185 190 Tyr Phe Arg Gly Gln Ser Cys Asn Ser GlnPro Leu Ser His Lys Val 195 200 205 Tyr Met Arg Asn Phe Lys Tyr Pro GlyAsp Leu Val Leu Met Glu Glu 210 215 220 Lys Lys Leu Asn Tyr Cys Thr ThrGly Gln Ile Trp Ala His Ser Ser 225 230 235 240 Tyr Leu Gly Ala Val PheAsn Leu Thr Val Ala Asp His Leu Tyr Val 245 250 255 Asn Ile Ser Gln LeuSer Leu Ile Asn Phe Glu Glu Ser Lys Thr Phe 260 265 270 Phe Gly Leu TyrLys Leu 275 <210> SEQ ID NO 7 <211> LENGTH: 235 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <400> SEQUENCE: 7 Met Ser Thr Glu Ser Met Ile ArgAsp Val Glu Leu Ala Glu Gly Pro 1 5 10 15 Leu Pro Lys Lys Ala Gly GlyPro Gln Gly Ser Lys Arg Cys Leu Cys 20 25 30 Leu Ser Leu Phe Ser Phe LeuLeu Val Ala Gly Ala Thr Thr Leu Phe 35 40 45 Cys Leu Leu His Phe Arg ValIle Gly Pro Gln Glu Glu Glu Gln Ser 50 55 60 Pro Asn Asn Leu His Leu ValAsn Pro Val Ala Gln Met Val Thr Leu 65 70 75 80 Arg Ser Ala Ser Arg AlaLeu Ser Asp Lys Pro Leu Ala His Val Val 85 90 95 Ala Asn Pro Gln Val GluGly Gln Leu Gln Trp Leu Ser Gln Arg Ala 100 105 110 Asn Ala Leu Leu AlaAsn Gly Met Lys Leu Thr Asp Asn Gln Leu Val 115 120 125 Val Pro Ala AspGly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Ser 130 135 140 Gly Gln GlyCys Arg Ser Tyr Val Leu Leu Thr His Thr Val Ser Arg 145 150 155 160 PheAla Val Ser Tyr Pro Asn Lys Val Asn Leu Leu Ser Ala Ile Lys 165 170 175Ser Pro Cys His Arg Glu Thr Pro Glu Glu Ala Glu Pro Met Ala Trp 180 185190 Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp 195200 205 Arg Leu Ser Thr Glu Val Asn Gln Pro Glu Tyr Leu Asp Leu Ala Glu210 215 220 Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 225 230 235<210> SEQ ID NO 8 <211> LENGTH: 434 <212> TYPE: DNA <213> ORGANISM: Homosapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (15)<223> OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (19) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (133) <223>OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (388) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (424) <223>OTHER INFORMATION: n equals to a, t, g, or c <400> SEQUENCE: 8tctacacaag gtacngacng ctaccctgag ccaacccagc tcctcatggg gaccaagtct 60gtatgcgaag taggtagcaa ctggttccag cccatctacc tcggagccat gttctccttg 120caagaagggg acnagctaat ggtgaacgtc agtgacatct ctttggtgga ttacacaaaa 180gaagataaaa ccttctttgg agccttctta ctataggagg agagcaaata tcattatatg 240aaagtcctct gccaccgagt tcctaatttt ctttgttcaa atgtaattat aaccaggggt 300tttcttgggg ccgggagtag ggggcattcc cacagggaca acggtttagc tatgaaattt 360ggggggccca aaatttcaca acttcatngt tgcccttact tgatgagaag tacttaactt 420gganaaaagg cttg 434 <210> SEQ ID NO 9 <211> LENGTH: 493 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (288) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (296) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (309) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (314) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (340) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (343) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (348) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (369) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (385) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (410) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (417) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (423) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (431) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (434) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (437) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (444) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (459) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (486) <223> OTHERINFORMATION: n equals to a, t, g, or c <400> SEQUENCE: 9 aattcggcagagaaattcca tactatcacc agttggccaa ctttccaagt ctagtgcaga 60 aatccaaggcacctcacacc tagagttcct atacctctga gactccagag gaaagaacaa 120 gacagtgcagaaggatatgt tagaacccac tgaaaaccta gaaggttaaa aaggaagcat 180 accctcctgacctataagaa aattttcagt ctgcaggggg atatccttgt ggcccaagac 240 attggtgttatcatttgact aagaggaaat tatttgtggt gagctccnag tgaggnttag 300 ggaccaggnggtgnccaagt ttctatcact tacctcatgn ctntaagnca agtgttttgt 360 tcccattgntgatggggtta aaacnttcag ccatcacttt tggggcaagn atggggnttt 420 gangggttggngcnggnctt gtcntcgtaa acagggggnt tggtgggttt ttctgggtcc 480 ttgggnaggactt 493 <210> SEQ ID NO 10 <211> LENGTH: 380 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (53)..(54) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (258) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (316) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (324) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (346) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (367) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (378) <223> OTHER INFORMATION: n equals to a, t, g, or c<400> SEQUENCE: 10 ggcagaggtt caatttgatc ataaatttgc ttcaattcaggagctttgaa ggnngtccaa 60 ggaaagctct agaaaacagt ataaactttc agaggcaaaatccttcacca atttttccac 120 atactttcat gccttgccta aaaaaaatga aaagagagttggtatgtctc atggaatgtt 180 cacacagaag gagttggttt tcatgtcatc tacagcatatgagaaaagct acctttcttt 240 tgattatgta cacaggtntc taaataagga agtatgagtttcacatgtat attcaaaaat 300 acaacagttg cttgtnttca gttngggttt ttcttggcccacccantttt ggtgctgggg 360 gttctanctt taaccccnga 380 <210> SEQ ID NO 11<211> LENGTH: 458 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (9) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (12) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (119) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (303) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (311) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (387) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (409) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (425) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (427) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (453) <223> OTHER INFORMATION: n equals to a, t, g, or c<400> SEQUENCE: 11 ggcacagcng gnagtagggg gcattccaca gggacaacggtttagctatg aaatttgggg 60 cccaaaattt cacacttcat gtgccttact gatgagagtactaactggaa aaaggctgna 120 agagagcaaa tatattatta agatgggttg gaggattggcgagtttctaa atattaagac 180 actggatcac tgaaatgaat ggatgatcta ctcgggtccaggattgaaag agaaatattt 240 caacaccttc ctgctataca atggtcacca gtggtccagttattgttcca atttggatcc 300 atnaatttgc nttcaattcc aggagctttg gaaggaattccaaggaaagc tccaggaaaa 360 ccgtattaaa ctttccaggg gccaaantcc ttcaccaattttttccacna actttccagg 420 cctgncncaa aaaaatggaa agggagttgg tangtccc 458<210> SEQ ID NO 12 <211> LENGTH: 388 <212> TYPE: DNA <213> ORGANISM:Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(11)..(12) <223> OTHER INFORMATION: n equals to a, t, g, or c <221>NAME/KEY: misc_feature <222> LOCATION: (46) <223> OTHER INFORMATION: nequals to a, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION:(50) <223> OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (81) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (138) <223>OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (155) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (182) <223>OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (188) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (269) <223>OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (317) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (322) <223>OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_difference <222> LOCATION: (358) <223> OTHER INFORMATION: n equalsto a, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (363)<223> OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (375) <223> OTHER INFORMATION: n equals toa, t, g, or c <400> SEQUENCE: 12 ctgcactggg nncatgaact aggcctggccttcaccaaga accgantgan ctataccaac 60 aaattcctgc tgatcccaga ntcgggagactacttcattt actcccaggt cacattccgt 120 gggaatgaac ctctgaantg ccagtgaaaatcagncaagc aggccgacca aacaagccag 180 antccatnca ctgtggtcat caccaaggtaacagacagct accctgagcc aacccagctc 240 cttcatgggg accaagtttg tttgcgaantaggttagcaa ctggttccag cccattttac 300 cttgggggcc agttctnctt gncaagaaggggacaagctt atggtggaac gttcatanca 360 tcntttttgg gtggntttac acaaaagg 388<210> SEQ ID NO 13 <211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM: Homosapiens <400> SEQUENCE: 13 gcgcggatcc accatgagac gctttttaag caaagtc 37<210> SEQ ID NO 14 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Homosapiens <400> SEQUENCE: 14 cgcgtctaga ctatagtaag aaggctccaa agaagg 36<210> SEQ ID NO 15 <211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM: Homosapiens <400> SEQUENCE: 15 gcgcggatcc accatgagac gctttttaag caaagtc 37<210> SEQ ID NO 16 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Homosapiens <400> SEQUENCE: 16 cgcgtctaga ctatagtaag aaggctccaa agaagg 36<210> SEQ ID NO 17 <211> LENGTH: 56 <212> TYPE: DNA <213> ORGANISM: Homosapiens <400> SEQUENCE: 17 cgctctagat caagcgtagt ctgggacgtc gtatggatagtaagaaggct ccaaag 56 <210> SEQ ID NO 18 <211> LENGTH: 733 <212> TYPE:DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 18 gggatccggagcccaaatct tctgacaaaa ctcacacatg cccaccgtgc ccagcacctg 60 aattcgagggtgcaccgtca gtcttcctct tccccccaaa acccaaggac accctcatga 120 tctcccggactcctgaggtc acatgcgtgg tggtggacgt aagccacgaa gaccctgagg 180 tcaagttcaactggtacgtg gacggcgtgg aggtgcataa tgccaagaca aagccgcggg 240 aggagcagtacaacagcacg taccgtgtgg tcagcgtcct caccgtcctg caccaggact 300 ggctgaatggcaaggagtac aagtgcaagg tctccaacaa agccctccca acccccatcg 360 agaaaaccatctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc 420 catcccgggatgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct 480 atccaagcgacatcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga 540 ccacgcctcccgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg 600 acaagagcaggtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc 660 acaaccactacacgcagaag agcctctccc tgtctccggg taaatgagtg cgacggccgc 720 gactctagaggat 733 <210> SEQ ID NO 19 <211> LENGTH: 1116 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <400> SEQUENCE: 19 atggccgagg atctgggactgagctttggg gaaacagcca gtgtggaaat gctgccagag 60 cacggcagct gcaggcccaaggccaggagc agcagcgcac gctgggctct cacctgctgc 120 ctggtgttgc tccccttccttgcaggactc accacatacc tgcttgtcag ccagctccgg 180 gcccagggag aggcctgtgtgcagttccag gctctaaaag gacaggagtt tgcaccttca 240 catcagcaag tttatgcacctcttagagca gacggagata agccaagggc acacctgaca 300 gttgtgagac aaactcccacacagcacttt aaaaatcagt tcccagctct gcactgggaa 360 catgaactag gcctggccttcaccaagaac cgaatgaact ataccaacaa attcctgctg 420 atcccagagt cgggagactacttcatttac tcccaggtca cattccgtgg gatgacctct 480 gagtgcagtg aaatcagacaagcaggccga ccaaacaagc cagactccat cactgtggtc 540 atcaccaagg taacagacagctaccctgag ccaacccagc tcctcatggg gaccaagtct 600 gtatgcgaag taggtagcaactggttccag cccatctacc tcggagccat gttctccttg 660 caagaagggg acaagctaatggtgaacgtc agtgacatct ctttggtgga ttacacaaaa 720 gaagataaaa ccttctttggagccttctta ctataggagg agagcaaata tcattatatg 780 aaagtcctct gccaccgagttcctaatttt ctttgttcaa atgtaattat aaccaggggt 840 tttcttgggg ccgggagtaggggcattcca cagggacaac ggtttagcta tgaaatttgg 900 ggcccaaaat ttcacacttcatgtgcctta ctgatgagag tactaactgg aaaaaggctg 960 aagagagcaa atatattattaagatgggtt ggaggattgg cgagtttcta aatattaaga 1020 cactgatcac taaatgaatggatgatctac tcgggtcagg attgaaagag aaatatttca 1080 acaccttcct gctatacaatggtcaccagt ggtcca 1116 <210> SEQ ID NO 20 <211> LENGTH: 251 <212> TYPE:PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 20 Met Ala Glu Asp LeuGly Leu Ser Phe Gly Glu Thr Ala Ser Val Glu 1 5 10 15 Met Leu Pro GluHis Gly Ser Cys Arg Pro Lys Ala Arg Ser Ser Ser 20 25 30 Ala Arg Trp AlaLeu Thr Cys Cys Leu Val Leu Leu Pro Phe Leu Ala 35 40 45 Gly Leu Thr ThrTyr Leu Leu Val Ser Gln Leu Arg Ala Gln Gly Glu 50 55 60 Ala Cys Val GlnPhe Gln Ala Leu Lys Gly Gln Glu Phe Ala Pro Ser 65 70 75 80 His Gln GlnVal Tyr Ala Pro Leu Arg Ala Asp Gly Asp Lys Pro Arg 85 90 95 Ala His LeuThr Val Val Arg Gln Thr Pro Thr Gln His Phe Lys Asn 100 105 110 Gln PhePro Ala Leu His Trp Glu His Glu Leu Gly Leu Ala Phe Thr 115 120 125 LysAsn Arg Met Asn Tyr Thr Asn Lys Phe Leu Leu Ile Pro Glu Ser 130 135 140Gly Asp Tyr Phe Ile Tyr Ser Gln Val Thr Phe Arg Gly Met Thr Ser 145 150155 160 Glu Cys Ser Glu Ile Arg Gln Ala Gly Arg Pro Asn Lys Pro Asp Ser165 170 175 Ile Thr Val Val Ile Thr Lys Val Thr Asp Ser Tyr Pro Glu ProThr 180 185 190 Gln Leu Leu Met Gly Thr Lys Ser Val Cys Glu Val Gly SerAsn Trp 195 200 205 Phe Gln Pro Ile Tyr Leu Gly Ala Met Phe Ser Leu GlnGlu Gly Asp 210 215 220 Lys Leu Met Val Asn Val Ser Asp Ile Ser Leu ValAsp Tyr Thr Lys 225 230 235 240 Glu Asp Lys Thr Phe Phe Gly Ala Phe LeuLeu 245 250 <210> SEQ ID NO 21 <211> LENGTH: 434 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (15) <223> OTHER INFORMATION: n equals to a, t, g, or c <221>NAME/KEY: misc_feature <222> LOCATION: (19) <223> OTHER INFORMATION: nequals to a, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION:(133) <223> OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (388) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (424) <223>OTHER INFORMATION: n equals to a, t, g, or c <400> SEQUENCE: 21tctacacaag gtacngacng ctaccctgag ccaacccagc tcctcatggg gaccaagtct 60gtatgcgaag taggtagcaa ctggttccag cccatctacc tcggagccat gttctccttg 120caagaagggg acnagctaat ggtgaacgtc agtgacatct ctttggtgga ttacacaaaa 180gaagataaaa ccttctttgg agccttctta ctataggagg agagcaaata tcattatatg 240aaagtcctct gccaccgagt tcctaatttt ctttgttcaa atgtaattat aaccaggggt 300tttcttgggg ccgggagtag ggggcattcc cacagggaca acggtttagc tatgaaattt 360ggggggccca aaatttcaca acttcatngt tgcccttact tgatgagaag tacttaactt 420gganaaaagg cttg 434 <210> SEQ ID NO 22 <211> LENGTH: 417 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (4) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (8) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (17) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (24) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (28) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (31)..(32) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (35) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (41)..(43) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (46) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (48) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (50) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (53) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (55) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (61)..(63) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (66)..(67) <223> OTHER INFORMATION: n equals to a, t, g,or c <221> NAME/KEY: misc_feature <222> LOCATION: (202) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (209) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (282) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (306) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (321) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (344) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (346) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (380)..(381) <223> OTHER INFORMATION: n equals to a, t,g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (395) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (405) <223> OTHER INFORMATION: n equals to a, t, g, or c<400> SEQUENCE: 22 attncggnac gagcagnggc atgnccgngg nnctnggactnnnctntngn gananagcca 60 nnnttnnaat gctgccagag cacggcagct gcaggcccaaggccaggagc agcagcgcac 120 gctgggctct cacctgctgc ctggtgttgc tccccttccttgcaggactc accacatacc 180 tgcttgtcag ccagcttcgg gnccagggng aggcctgtgtgcagttccag ggtctaaaag 240 gacaggagtt tgcaccttca catcagcaag tttatgcacctnttagagca gacggagata 300 agccangggg acaactgaca nttgtgagac aaattccacacagnanttta aaatcagttt 360 ccagttttga atggggacan nattaggctg gcttnacaagaccgntggat tttacag 417 <210> SEQ ID NO 23 <211> LENGTH: 388 <212> TYPE:DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (11)..(12) <223> OTHER INFORMATION: nequals to a, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION:(46) <223> OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (50) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (81) <223>OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (138) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (155) <223>OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (182) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (188) <223>OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (269) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (317) <223>OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (322) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (358) <223>OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (363) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (375) <223>OTHER INFORMATION: n equals to a, t, g, or c <400> SEQUENCE: 23ctgcactggg nncatgaact aggcctggcc ttcaccaaga accgantgan ctataccaac 60aaattcctgc tgatcccaga ntcgggagac tacttcattt actcccaggt cacattccgt 120gggaatgaac ctctgaantg ccagtgaaaa tcagncaagc aggccgacca aacaagccag 180antccatnca ctgtggtcat caccaaggta acagacagct accctgagcc aacccagctc 240cttcatgggg accaagtttg tttgcgaant aggttagcaa ctggttccag cccattttac 300cttgggggcc agttctnctt gncaagaagg ggacaagctt atggtggaac gttcatanca 360tcntttttgg gtggntttac acaaaagg 388 <210> SEQ ID NO 24 <211> LENGTH: 458<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (9) <223> OTHER INFORMATION: nequals to a, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION:(12) <223> OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (119) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (303) <223>OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (311) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (387) <223>OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (409) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (425) <223>OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (427) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (453) <223>OTHER INFORMATION: n equals to a, t, g, or c <400> SEQUENCE: 24ggcacagcng gnagtagggg gcattccaca gggacaacgg tttagctatg aaatttgggg 60cccaaaattt cacacttcat gtgccttact gatgagagta ctaactggaa aaaggctgna 120agagagcaaa tatattatta agatgggttg gaggattggc gagtttctaa atattaagac 180actggatcac tgaaatgaat ggatgatcta ctcgggtcca ggattgaaag agaaatattt 240caacaccttc ctgctataca atggtcacca gtggtccagt tattgttcca atttggatcc 300atnaatttgc nttcaattcc aggagctttg gaaggaattc caaggaaagc tccaggaaaa 360ccgtattaaa ctttccaggg gccaaantcc ttcaccaatt ttttccacna actttccagg 420cctgncncaa aaaaatggaa agggagttgg tangtccc 458

What is claimed is:
 1. An isolated polypeptide consisting of an aminoacid sequence selected from the group consisting of: (a) amino acidresidue −27 to amino acid residue +147 as set forth in SEQ ID NO:2; (b)amino acid residue −26 to amino acid residue +147 as set forth in SEQ IDNO:2; (c) amino acid residue +1 to amino acid residue +147 as set forthin SEQ ID NO:2; (d) a full-length polypeptide having the amino acidsequence expressed by the cDNA plasmid contained in ATCC Deposit No.75927; (e) a full-length polypeptide, excluding the N-terminalmethionine residue, having the amino acid sequence expressed by the cDNAplasmid contained in ATCC Deposit No. 75927; and (f) a polypeptidehaving the amino acid sequence, excluding the signal sequence, encodedby the cDNA plasmid contained in ATCC Deposit No.
 75927. 2. The isolatedpolypeptide of claim 1 consisting of amino acid residue −27 to aminoacid residue +147 as set forth in SEQ ID NO:2.
 3. The isolatedpolypeptide of claim 1 consisting of amino acid residue −26 to aminoacid residue +147 as set forth in SEQ ID NO:2.
 4. The isolatedpolypeptide of claim 1 consisting of amino acid residue +1 to amino acidresidue +147 as set forth in SEQ ID NO:2.
 5. The isolated polypeptide ofclaim 1 consisting of a full-length polypeptide having the amino acidsequence expressed by the human cDNA contained in ATCC Deposit No.75927.
 6. The isolated polypeptide of claim 1 consisting of afull-length polypeptide, excluding the N-terminal methionine residue,having the amino acid sequence expressed by the human cDNA contained inATCC Deposit No
 75927. 7. The isolated polypeptide of claim 1 consistingof a polypeptide having the amino acid sequence expressed by arecombinant cell comprising the human cDNA contained in ATCC Deposit No.75927.
 8. The isolated polypeptide of claim 1 wherein said polypeptideis fused to a heterologous polypeptide.
 9. The isolated polypeptide ofclaim 8 wherein said heterologous polypeptide is the Fc domain ofimmunoglobulin.
 10. A composition comprising the polypeptide of claim 1and a pharmaceutically acceptable carrier.
 11. An isolated polypeptideencoded by a nucleic acid molecule consisting of a polynucleotidesequence selected from the group consisting of: (a) a polynucleotidesequence consisting of at least 30 contiguous nucleotides of nucleotides783 to 1304 of SEQ ID NO:1; and (b) a polynucleotide sequence consistingof at least 30 contiguous nucleotides of the open reading frame encodedby the cDNA plasmid contained in ATCC Deposit No.
 75927. 12. Theisolated polypeptide of claim 11 encoded by a polynucleotide whichconsists of (a).
 13. The isolated polypeptide of claim 11 encoded by apolynucleotide which consists of (b).
 14. The isolated polypeptide ofclaim 11 wherein said polypeptide is fused to a heterologouspolypeptide.
 15. The isolated polypeptide of claim 14 wherein saidheterologous polypeptide is the Fc domain of immunoglobulin.
 16. Acomposition comprising the polypeptide of claim 11 and apharmaceutically acceptable carrier.
 17. An isolated polypeptideconsisting of an amino acid sequence selected from the group consistingof: (a) an amino acid sequence consisting of at least 30 contiguousamino acid residues of SEQ ID NO:2; and (b) an amino acid sequenceconsisting of at least 30 contiguous amino acid residues encoded by thecDNA plasmid contained in ATCC Deposit No.
 75927. 18. The isolatedpolypeptide of claim 17 which consists of (a).
 19. The isolatedpolypeptide of claim 17 which consists of (b).
 20. The isolatedpolypeptide of claim 17 wherein said polypeptide is fused to aheterologous polypeptide.
 21. The isolated polypeptide of claim 20wherein said heterologous polypeptide is the Fc domain ofimmunoglobulin.
 22. A composition comprising the polypeptide of claim 17and a pharmaceutically acceptable carrier.